Methods and compositions for analyte detection

ABSTRACT

The present invention is directed to methods and apparatus for detection of one or more analytes. Analytes include agents or components of infectious agents such as pathogenic virus, as well as enzymes, proteins and biomarkers.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.60/775,649, filed Feb. 21, 2006, No. 60/789,345, filed Apr. 5, 2006, andNo. 60/866,932 filed Nov. 22, 2006, pursuant 35 U.S.C. 119(e), and thedisclosure for each of which is incorporated by reference herein in itsentirety.

STATEMENT AS TO GOVERNMENT SUPPORTED RESEARCH

Portions of this invention may have been made with the support of theUnited States government under contract number 200-2007-19345 granted bythe Center for Disease Control. The Government may have certain rightsto portions of this invention.

BACKGROUND OF THE INVENTION

This invention relates to assays for analyte(s), e.g., antigens, in asample such as a biological sample obtained from an animal. Inparticular, the invention relates to a method and device(s) for thedetection of an analyte(s) utilizing binding moieties specificallytargeting a selected analyte. More particularly, the analytes to bedetected include infectious agents and/or components thereof.

Many types of assays have been used to detect the presence of varioussubstances, often generally called analytes or ligands, in bodilysamples. These assays typically involve antigen antibody reactions,ligand, anti-ligand, ligand receptor and utilize, synthetic conjugatescomprising radioactive, enzymatic, fluorescent, or visually observablemetal soluble tags, and specially designed reactor chambers. Mostcurrent tests are designed to make a quantitative determination, but inmany circumstances all that is required is qualitative orpositive/negative indication. Assays have been utilized to detectinfectious agents, such as influenza.

Even the positive/negative assays must be very sensitive because of theoften small concentration of the analyte of interest in the test fluid.False positives can also be troublesome, particularly with agglutinationand other rapid detection methods such as dipstick and color changetests. Because of these problems, sandwich assays and other sensitivedetection methods which use metal sols or other types of coloredparticles have been developed. These techniques have not solved all ofthe problems encountered in these rapid detection methods. Moreover,with the emergence of highly pathogenic agents such as influenza virus,there is a need to develop effective laboratory or point-of-care methodsand systems that can effectively and accurately detect one or moreinfectious agents, such as influenza Types or strains within subtypes.

Influenza is commonly seen in local outbreaks or epidemics throughoutthe world. Epidemics may appear at any time and can occur explosivelywith little or no warning. The number of people affected can vary from afew hundred to hundreds of thousands. Epidemics may be short-lived,lasting days or weeks but larger epidemics may last for months. Althoughinfluenza is a mild disease in most individuals, it is life threateningto elderly, the very young or debilitated individuals. Epidemics areresponsible for large losses in productivity. Therefore, there is a needto develop devices and methods to effectively detect what Types andsubtypes of a pathogen, such as influenza, present in samples obtainedfrom subjects in order to determine whether the infection is caused by atypical or expected subtype of Influenza (seasonal flu) or a subtypethat is the causative agent of an epidemic or pandemic.

It is an object of this invention to provide a rapid, sensitive methodfor detecting analytes in a biological sample. Another object is toprovide an assay which has high sensitivity and fewer false positivesthan conventional assays. A further object is to provide an apparatus orsystem for detection of low levels of analytes present in biologicalsamples. Another object is to provide an assay system which involves aminimal number of procedural steps, and yields reliable results evenwhen used by untrained persons. An additional object is to provide asystem for testing infectious agents that provides results identifyingone or more infectious agents in a matter of minutes. A further objectprovides a system where results on a testing implement are equallyspecific and sensitive for the target analytes, notwithstanding thatresults can be read one to several hours after completion of a reactionnecessary to obtain a result. These and other objects and features ofthe invention will be apparent from the following description, drawing,and claims.

SUMMARY OF THE INVENTION

In certain aspects of the invention a sample collection device (SCD) isprovided for use in detection of one or more target antigens or analytesthat may be present in a sample. The sample collection device can beutilized in conjunction with a test device. In one aspect, a system isprovided for detection of one or more analyte comprising a SCD, a TestDevice and a Reader, as further described herein.

In various embodiments, a SCD comprises one or more upper sealedchambers, which can contain the same or different solutions. In oneembodiment, the upper sealed chamber comprises at least two compartmentsor subchambers each comprising. In other embodiments, the upper chambercan comprise puncturable, breakable or rupturable ampoules. In someembodiments, a SCD provides the necessary reagents to form a complexwith one or more different target antigens that may be present in asample, wherein the complex comprises a capture moiety and a detectablelabel, a Test Device provides the necessary means to addressably captureone or more complexes so formed and a Reader which provides a means todetect one or more signals from addressably captured complexes.

In various embodiments, an upper sealed chamber comprising extractionbuffer and/or reagents, a sample collection implement, a samplecollection implement holder and a plurality of reagents, wherein thereagents comprise a plurality of specific binding pairs, where each paircomprises a label conjugated to first specific binding agent and acapture moiety conjugated to a second specific binding agent, where thefirst and second specific binding agents specifically bind a targetantigen to form a complex. The capture moiety can be “captured” orimmobilized where a partner capture moiety disposed on a substrate bindsto the capture moiety-specific binding agent conjugate.

In various embodiments, the plurality of specific binding agentscomprise a multitude of groups of specific binding pairs, wherein eachgroup comprises specific binding agents that specifically bind onetarget antigen, and a second group of specific binding pairsspecifically bind a second different antigen. Thus a plurality ofspecific binding pairs comprised in a SCD is capable of detecting aplurality of different antigens.

In various embodiments, the capture moiety is an oligonucleotide,avidin, streptavidin, pyranosyl RNA (pRNA), aptamer, or a combinationthereof. In various embodiments, the label is a metal, a fluorophore, achromophore or a combination thereof. In some embodiments, the pluralityof specific binding pairs comprised in a SCD can contain one type ofcapture moiety but with different capture moiety partners, e.g., eachspecific binding agent conjugated to pRNA, where each group that isspecific to a different antigen comprises different pRNAs. In otherembodiments, the plurality of specific binding pairs comprises one ormore different capture moieties, e.g., pRNA for one group of specificbinding pairs, while streptavidin for another, or a combination ofdifferent types of capture moieties.

In some embodiments, the plurality of specific binding pairs comprisedin a SCD can contain one type of label (e.g., specific binding pairswhere each group is conjugated to fluorophores having the same orfluorophores having different wavelength signals). In other embodiments,specific binding pairs can comprise a combination of different types oflabels (e.g., combination of metals and fluorophores). In oneembodiment, the capture moiety is pRNA and the label is Europium.

In various embodiments, the specific binding agents are antibodies, thusa specific binding pair comprises an antibody-label conjugate (“labelprobe”) or antibody-capture moiety (“capture probe”). In suchembodiments, a “partner capture moiety” is comprised on a test membranedisposed in a Test Device, which partner binds a specific capture probe,e.g., pRNA partner specifically binding a pRNA (i.e., capture moiety)contained on a capture probe (i.e., antibody specific for a targetantigen).

In some embodiments, the different analytes detected are virus orcomponents of virus (e.g., polypeptides). In various embodiments, thedifferent antigens are from influenza virus and subtypes of influenzavirus. In one embodiment, the influenza virus that can be detected isinfluenza virus A and B as well as subtypes of influenza virus. Oneembodiment is directed to detection of influenza virus strains A and Band subtypes of the formula HxNy, wherein x can be 1-16 and y can be1-9, or any combination of xy thereof.

In yet other embodiments, the different analytes detected are one ormore different infectious agents and/or one or more different subtypesof an infectious agents. Such infectious agents include HIV, HCV, andmyobacterium, tuberculosis, bacteria, fungi, yeast, HSV, HPV or acombination thereof.

In various embodiments, an SCD comprises a sampling implement thatprovides a means to collect a sample from a subject, wherein thesampling implement is comprised connected to the upper chamber via asampling implement holder. The sampling implement is disposed at thedistal end of a shaft, which shaft can be solid, hollow orsemi-permeable. In some embodiments, the sampling implement is a swab, acomb, a brush, a spatula, a rod, a foam, a flocculated substrate or aspun substrate.

In various embodiments, an SCD comprises one or more sealed upperchambers wherein the seal functions as a valve to control fluidcommunication between the upper chamber and lower chamber of an SCD. Insome embodiments, the valve can be a break-away valve, a flapper valve,a twist, screw, rupturable, puncturable or breakable valve. In otherembodiments, the upper chamber can contain one or more ampoules whichprevent solutions contained therein to flow to the lower chamber, unlesspressure is exerted to rupture, puncture or break the ampoule so as torelease any contents therein.

One aspect of the invention is directed to a SCD comprising a samplereservoir upstream of a plunger implement, a plurality of sealableapertures for delivery of one or more solutions, a substrate forfiltering one or more compounds from a sample administered to the samplereservoir and reagents that are capable of specifically binding at leastone analyte in said sample.

In another aspect of the invention, a Test Device is provided fordetection of one or more analytes, wherein the device comprises alateral flow membrane in a body, a chamber upstream of the lateral flowmembrane containing a fluid or solution, wherein a gap is disposedbetween said chamber and said lateral flow membrane thus precludingfluid communication between the chamber and the lateral flow membrane.In one embodiment, the pressure exerted on the chamber pushes close thegap thus forming fluid communication between the chamber and the lateralflow membrane. In one embodiment, an opening into which a distal end ofan SCD fits, is disposed directly above a wicking pad that is disposeddownstream of the gap, but upstream of the lateral flow membrane.

In one embodiment, the Test Device chamber comprises one or moresubchambers containing the same or different solutions. In otherembodiments, the chamber of subchambers comprise one or more ampoulesthat are breakable, puncurable or rupturabie. Thus, where pressure isexerted on such ampoules the contents are controllably released. Asdescribed herein, a Test Device can comprise a gap means or not comprisea gap means for disrupting fluid communication from the chamber to thelateral flow membrane. A Test Device gap can be from zero to 3.0, 0.5,to 3.5, 1.0 to 2.5, 1.0 to 3.0, or 2.0 to 4.0 mm.

In some embodiments, a Test Device can comprise a body housing thelateral flow membrane, wherein the body provides one or a plurality ofwindows through which the lateral flow membrane is visible. In variousembodiments described herein, a Test Device comprises a lateral flowmembrane that comprises a wicking substrate and an absorbent substrateupstream or downstream of the test zones disposed on said lateral flowmembrane. In some embodiments, a substrate for collecting a small volumeof sample for archiving is provided in a SCD or Test Device. In oneembodiment, the substrate providing such archiving means is a filter,membrane or paper that collects a small volume of sample and saidsubstrate is subsequently removed from the device.

In various embodiments, an SCD and/or Test Device comprises one or moreidentical identifiable tags, which can be removed from one device andplaced on another device.

In some embodiments, the Test Device is shaped to only fit (specializedadaptor shape) into the receiving port of a reader if the upstreamchamber has been depressed thus indicating that wash buffer or chasebuffer contained therein has been released through the lateral flowmembrane. In such embodiments, the Test Device and Reader specializedadaptor provides a means to verify that chase buffer or solution in theupstream chamber of the Test Device has been released and thus washedany sample present upstream of the lateral flow membrane through thelateral flow membrane. Thereby, the specialized adaptor provides a“safety means” to prevent reading of unprocessed samples.

In another aspect of the invention, the processed samples are runthrough the Test Device's lateral flow membrane, but can be placed asidefrom 30 minutes to several hours. In various embodiments, a plurality ofsamples can be run through the Test Device but read at 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11 or 12 hours later, with consistent and accuratesignals.

In certain aspects of the invention, the devices disclosed herein areutilized in methods for detection of one or more analyte that may bepresent in a sample. In some embodiments, methods are directed todetecting one or more strain of an infectious agent. In one embodiment,a method is directed to utilizing the devices of the invention to detectone or more influenza virus and subtypes thereof. For example, methodsare provided for detection of influenza virus A and B, and subtype ofinfluenza A that may be present in a single sample.

In one embodiment, a method is provided for determining whether asubject is infected with a pandemic, non-pandemic or strain of influenzavirus for which vaccine is available.

In some embodiments, the Test Device excludes any reagent or bindingagent that is capable of specifically binding a target antigen.

In one aspect of the invention, a reader is providing to detect a signalfrom a Test Device wherein said reader is a UV LED reader. In variousembodiments, the signal detected is a fluorescence signal from adetector molecule. In further embodiments, the detector molecule is alanthanide. In yet a further embodiment, the lanthanide is Europium.

In one embodiment, the reader comprise a UV photodiode. In anotherembodiment, the reader comprises a UV laser diode.

In another aspect of the invention, a reader is configured to compriseat least one hard standard. In another embodiment, a reader isconfigured to comprise at least two or more hard standards. In variousembodiments, a hard standard comprises a label molecule emitting adetectable signal. In further embodiments, the label is a fluorescencelabel. In yet further embodiments, the fluorescence label is alanthanide. In yet a further embodiment, the lanthanide is Europium.

In another aspect of the invention, an SCD and Test device of theinvention are used in a method to detect one or more analytes, whereinsuch an analyte is associated with a disease, pathologic or otherphysiological condition. In various embodiments, such analytes arebiomarkers associated with a condition related to the heart, liver,kidney, intestine, brain, fetus, or pancreas. In one embodiment, suchanalytes are associated with a cardiac condition (e.g., myocardialinfarction).

In various embodiments, the devices of the invention can be utilized inany method to detect an antigen or protein (analytes) in a sampleobtained from a subject to detect any such analytes, through utilizationof a particular panel of immunoreactive or specific binding reagentsthat are specific for the desired analytes.

In several aspects of the invention, the Test Device comprises anupstream chamber that contains a means for providing a wash/runningbuffer or liquid. In various embodiment, such a buffer or liquidcomprises additional agents such as signal/detector molecules that canbe read by an optical reader or by unaided visualization. In certainembodiments, the buffer or liquid is comprised in a compartment thatcomprised of a glass ampoule or membrane pouch, sac or formed filledpouch. In further embodiments, such compartments are ruptured, broken orotherwise released of their contents by exerting pressure on saidcompartments. In other embodiments, such compartments are punctured orlanced by an appendage or needle. In yet further embodiments, suchcompartments are protected by a safeguard means that precludesaccidental or unintentional release of their contents.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates a sample collection device.

FIG. 2A illustrates a sample collection device with a blow up view of amixing compartment.

FIG. 2B illustrates a sampling assembly.

FIG. 3 illustrates a test device.

FIG. 4 illustrates a reader.

FIG. 5 illustrates a sample collection device.

FIG. 6 illustrates a sample collection device.

FIG. 7 illustrates a test device comprising a gap means.

FIG. 8 illustrates a sample collection device and test device.

FIG. 9A and 9B illustrates test strips tested with different volume ofextraction buffer.

FIGS. 10A and 10B illustrate effects of background clean-up and variousamounts of extraction buffer.

FIG. 11 illustrates two different tests for detection of influenza A.

FIG. 12 illustrates two different tests for detection of influenza A andinfluenza B.

FIG. 13 illustrates the detection of influenza A using gold as label.

FIG. 14 illustrates the detection of influenza B using gold as label.

FIG. 15 illustrates the detection of influenza A using Europiumconjugate.

FIG. 16 illustrates the detection of influenza B using Europiumconjugate.

FIG. 17 illustrates sensitivity of Europium conjugate in detection ofinfluenza B.

FIG. 18 illustrates strips tested with influenza A subtypes.

FIG. 19 illustrates buffer only control.

FIG. 20 illustrates test for influenza A and subtype H3N2.

FIG. 21 illustrates test for influenza B.

FIG. 22 illustrates test for influenza A and subtype H5N1.

FIG. 23 illustrates test for influenza A and subtype H1N1.

FIG. 24 illustrates test for influenza B and influenza A subtype H5N1.

FIG. 25 illustrates test for Epstein-Barr virus

FIG. 26 illustrates a test device comprising multiple test lines fordifferent type and subtypes of an analyte; 901 filter or sample pad ontowhich a sample is applied; 902 reagent pad; 903 test line for type 1 ofan analyte/infectious agent; 904 type 2 of an analyte/infectious agent;905 subtype of type 1 or type 2; 906 subtype of type 1 or type 2; 907control line.

FIG. 27 illustrates various aspects of one Test Device; (A) an assembledTest Device 1001; (B) an unassembled Test Device with upper 1002, lower1004 and test strip 1003, as well as the aperture/port to receive asample 1005; (c) closer view of the spring/button compartment which canbe pushed down to rupture, break or puncture a buffer/liquid compartmentdisposed above the membrane/pad 1006 that is in fluid communication withthe test strip 1003.

FIG. 28(A) illustrates an embodiment wherein a collar lock is providedthat connects an SCD 1101 and Test Device 1105 component, wherein thecollar lock 1104 provides channels/pores 1103 in a geometricalconfiguration that is open to an upstream compartment 1102 only whenturned in the lock position (i.e., allowing fluid flow from the upstreambuffer/liquid compartment laterally through the Test Device. Theupstream compartment 1102 can be an ampoule or formed filled sac.

FIG. 28(B) illustrates an embodiment of a Test Device whereby the devicecomprises a safeguard cover 1107 over the upstream compartment 1106containing a liquid (e.g., wash/running buffer). Furthermore, the devicecomprises a reservoir 1109 in fluid communication with the sampleaperture/port 1108 which reservoir can comprise a membrane orcompartment for retaining an archive sample.

FIG. 28: (C) illustrates a compartment (e.g., ampoule) 1111 disposed ina Test Device with a break point 1110, wherein the Test Device isconfigured to provide a trough/channel 1112 and supporting substrate1113 which directs the liquid contents of the ampoule downstream to thetest strip; (D) provides an overhead view of the compartment comprisinga sac/ampoule 1114 positioned upstream of where sample introductionoccurs 1115, configured to provide a fluid ramp 1119 and supports 1118through which the contents of the compartment to a space 1117 that is influid communication with a wicking pad 1116.

FIG. 29 provides one illustration of an sampling device with an archivalcomponent; the sampling swab 1205 is attached to a hollow shaft 1201 andis flanked by sleeves 1202 upstream of a hydrophobic frit (10+u) 1206and 1203 followed by a (FIG. 29B) filter paper that can be in variousshapes with regions for retaining an archive sample 1209 and threedimensional regions 1210 that can for example retain a reagentpill/bead. In addition, reagent pills/beads can also be disposed in thecompartment downstream of the filter paper 1208 which can allow mixingbefore expelling the sample through the tip 1204.

FIG. 30 illustrates (A) striped test strip results utilizing pRNA and(B) monoclonal antibodies.

FIG. 31 illustrates a comparison of sensitivity using pRNA and antibody.

FIG. 32 provides a schematic of pRNA binding of multiple analytes on atest strip.

FIG. 33. Hard Standards; (A) graph of six different concentrations ofEuropium as read of an UV LED reader; (B) photo of the six hardstandards; (C) graph for concentration #1; (D) graph for concentrationof #5; (E) graph for concentration #3; (F) graph for concentration of#6; (G) graph for concentration of #2; (H) graph for concentration of#4.

FIG. 34. Hard Standards; (A) provides a graph for 7 different hardstandards as read by an UV LED reader; (B) provides a Test Device withmultiple windows through which the signal is detected when the TestDevice is placed in a reader.

FIG. 35 illustrates an embodiment where an upstream compartment 1401(e.g., ampoule) containing a liquid is in fluid communication with a drywicking pad 1402 which swells when said compartment is manipulated torelease its liquid content, wherein said swelling increases the size ofthe wicking pad 1403 so that it becomes in fluid communication with asample 1405 disposed on a test strip 1406. Therefore, based on thedensity/type of wicking material 1402 a predetermined time delay can bebuilt into the system from the point of release to point of initiatingflow of the sample 1405 through the test strip 1406.

FIG. 36 illustrates fluorescence intensity of subtype H5N1 viruses.

Figure depicts a graph of fluorescence intensity of subtype H1N1viruses.

FIG. 38 depicts a graph of fluorescence intensity of Flu B viruses.

FIG. 39 illustrates the sensitivity using a MAB system.

FIG. 40 illustrates the sensitivity using a pRNA system.

FIG. 41 illustrates the sensitivity using a pRNA system and membraneblocking.

FIG. 42 provides a comparison of sensitivity for MAB, pRNA and pRNA withblocking.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Various aspects of the present invention are directed to devices andbinding-pair assays that utilize specific binding moieties and capturemoieties for the qualitative and/or quantitative analysis of selectedanalytes in samples. The invention is useful in a variety of assays thatare utilized to detect one or more infectious agents present in asample. Assays useful in the invention include, but are not limited to,competitive immunoassays, non-competitive immunoassays, sandwichimmunoassays and blocking assays.

However, the use of the invention is not limited to immunoassays, asother assays including ligand-receptor, ligand-binding protein,aptamer-binding agent, enzyme-substrate, chemical or biochemicalreactions are detected by utilizing a device or method of the invention.In one aspect of the invention, methods and systems of the inventionutilize a sample collection device, a test device and a reader. Variousspecific assay protocols, reagents, devices or analytes useful in thepractice of the invention are known, see, e.g., U.S. Pat. Nos.4,313,734; 4,366,241; 5,266,266; 6,235,539; 6,468,474; 6,565,808;6,448,001; or 5,415,994, as well as U.S. Pub. Nos. 2006/0040405;2004/0014094; 2004/0048395; or 2005/0130120.

In one aspect of the invention, a sample collection device is utilizedto collect a sample and process a sample with immunoreactive reagentsthat provide a detection means and a capture means, which processedsample is subsequently provided to a test device, which provides a meansfor reading/viewing signals provided by said detection means. Thesignals can be read by the naked eye or with an instrument depending onthe combination of detection means (e.g., conjugate labels) utilized.Furthermore, the test device can be configured to allow detection ofmultiple analytes. Such analytes can be from one or more infectiousagents, including different strains and/or subtypes. Detection caninclude qualitative and/or quantitative measurements of one or moreanalytes.

Sample Collection Device. One aspect of the invention is directed to asample collection device (“SCD”) that comprises the necessary means tocollect a biological sample, as well as the reagents and buffersnecessary to process the sample and react to the binding reagents withone or more target analytes. As shown in FIG. 2B, an exemplary SCDcomprises an upper chamber 210 to which is attached a sampling implementholder 209 a stem 208 a sampling implement 207, collectively forming asampling assembly-.

The sampling assembly FIG. 1 is removable from a housing comprising asample receiving tube 103, a lower chamber mixing or reagent area 104,which can contain reagents that specifically bind to one or more targetantigens. The lower chamber 104 can comprise one or more compartments.For example, two compartments can be arranged in series in the lowerchamber. The sampling assembly is placed into the sample receiving tube103 portion of the housing to provide an integrated configuration. Insuch a configuration a sampling implement 107 is upstream of and influid communication with the lower chamber. The length the samplingassembly can be optimized for sample collection, e.g., throat and nasalsample collection. For example, the length of the device (e.g.,integrated configuration) can be about 1 to 9 inches, or about 3, 4, 5,6, 7, 8 or 9 inches.

The stem 102 can be hollow, solid or semi-porous. Therefore, in someembodiments, the stem actually provides a path of fluid communicationfrom the upper chamber 100 to the sampling implement 107 (e.g., swab).The stem is held by the sample holder 101 which fits into a receivingend of the upper chamber 100. For example, the stem may be hollow orsemi-permeable and a portion of the stem which may extend into the upperchamber has a terminal end that is closed, so that if the stem portionin the upper chamber is snapped or broken, then fluid communication(i.e., fluid flow) is provided between the upper chamber down throughthe distal end of the stem to a sampling implement (e.g., swab)comprising a sample (e.g., biological sample).

An upper chamber can comprise one or more compartments-. The upperchamber can be comprised of a semi-rigid or depressible material, andshaped as a bulb 99. Such a bulb can comprise a solution.

The upper chamber can be sealed. Furthermore such a seal can bepunctured, broken or opened via a valve structure, so as to providefluid communication between the upper chamber and lower chamber. In someembodiments, the solution in the upper sealed chamber is a buffersolution. In various embodiments, the volume for a solution in the upperchamber is from about 10-500 μl, or from about 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or500. In one embodiment, the solution volume is up to 150 μl.

In some embodiments, a liquid solution comprising the necessary reagents(e.g., detection/capture specific binding agents, etc.) can be disposedin the reagent area of the lower chamber in liquid communication withthe upper chamber. As exemplified in FIG. 2A, fluid from the upperchamber flows down to the sampling implement to extract sample and—theextracted sample passes through an aperture that may restrict/controlthe liquid flow from the upper chamber to the lower chamber comprising,for example, an aperture to control flow by size, e,g., size ofperforations or type of substrate or filter that is disposed on theproximal end 201 of a compartment in the lower chamber-. The lowerchamber may contain a reagent area. In one embodiment, the reagent area(e.g., 203) contains a solid substrate that includes the necessaryreagents 202 (e.g., immunoassay reagents, such as detection and captureprobes, etc.), formed as a dried solid, separately disposed or in aunified solid.

Therefore, where a sample is washed downward via the solutions (e.g.,buffer) in the upper chamber, a mixture is produced carrying the samplethat travels down to the lower chamber reagent area, which chambercomprises the solid reagent-containing substrate 202. The solid reagentis dissolved rapidly by the buffer and the resultant solution is amixture of sample that may contain analyte(s) of interest, and theimmunoassay reagents (e.g., specific binding agents, label conjugate andcapture probes, etc.). For example, a solid reagent 202can include bothlabel and capture probes used in the assay that are capable ofspecifically binding a target analyte. The integrated device 200includes the upper and lower chambers, the sample collection device andthe luer lock 206 which locks into the lateral flow device for deliveryof the reaction mixture for subsequent detection. Furthermore, as notedthe upper chamber can be designed with a depressible (e.g., plastic)bulb 205 so that if desired additional pressure is applied to forcefluid flow from an upper chamber into a lower chamber.

In one embodiment, the upper chamber comprises a valve that allowscontrollable release of a solution comprised in the upper chamber. Forexample, where the valve is a snap-valve, the user applies force to thevalve stem to break the stem, whereby the breakaway feature allowsbuffer to enter the lower chamber via the stem. Furthermore, the sealedchamber can be a squeezable bulb, which is capable of being compressed(e.g., user applies pressure to the bulb), thus controlling the flowrate of the solution (e.g., buffer) to the sampling implement.

In some embodiments, the upper chamber is comprised of a bulb componentthat is a self-contained compartment that includes a solution. Suchsolutions include extraction, lysis, reagent, buffer or preservativesolutions. In one embodiment, the solution is a buffer solution that isutilized to transfer the biological sample from the sampling implementdown to the lower chamber.

Furthermore, such an upper chamber can comprise one or morecompartments, such as depicted in FIG. 5. Each compartment can comprisea solution that is the same or different 501 and 502.

Furthermore, such solutions can comprise reagents as desired includingbut not limited to extraction buffers, reducing agents, immunoreactiveagents, such as, anti-analyte specific binding agents comprisingdetection labels (e.g., conjugates) and capture moieties. The reagentsfor reactions are depicted in 503 and 504 shows attachment of the bierto the test strip 505.

In another embodiment, the sampling assembly is not integrated with thehousing containing a sample receiving tube. See FIG. 6. In such aconfiguration, the sampling assembly 601 is utilized to collect anddeliver a sample to a sample receiving chamber 609. The sample receivingchamber can be open or closed to allow a sample to be introduced intosample receiving tube. It should be understood that any sample receivingtube disclosed herein can be of a variety of geometric shapes, includingcylinder, square, triangular or any polygonal, as desired. In someembodiments, the housing can comprise one or more sealable apertures 603that can be opened to add one or more selected reagents 607, buffers orwash fluids.

For example, in one embodiment, whole blood is drawn into the samplereceiving chamber 609. Subsequently, the sample passes through amembrane 602 (e.g., a membrane to separate red blood cells from plasma,allowing the plasma to pass through) into a lower portion of the samplereceiving tube to mix with various reagents, for example, necessary foran immunoassay. Immunoreagents—necessary to target specific analytes canbe pre-selected and disposed as a solid substrate 607 in the SCD oradded through an aperture 603, or can be disposed on a membrane 604.

As the whole blood sample is discharged, the membrane 604 may act as afilter to precludes passage of blood components, thus allowing onlyplasma to pass through the distal end of the sample receiving tube 605,which will fit into the Test Device 606. Additional valves that can beutilized include a rotary, breakable, stopcock, gate, ball, flapper,needle, butterfly, pinch, bellows, piston, slide, plug, diverter, oractuator valve.

As used herein, a “capture probe” refers to a conjugate of a bindingagent linked to a capture moiety and a “detection probe” refers to aconjugate of a binding agent linked to a label or signal producingmoiety, wherein each is capable of specifically binding to a targetanalyte. Furthermore, for clarity, a “capture moiety partner” in thecontext of the Test Device (described below) refers to a complement,cognate or partner molecule that specifically binds to a capture moietycomprised on a capture probe.

Solid reagent components include, a powder, pill, bead, lyophilizedpellet, pressed lyophilized power, dried on solid support (e.g.,glass/plastic bead), lyophilized on or in association with a solidsupport or dried directly in the mixing or lower chamber. Such reagentsare known in the art such as disclosed in CURRENT PROTOCOLS INIMMUNOLOGY (Coligan, John E. et. al., eds. 1999).

In some embodiments, as the solution passes through the samplingimplement, an extraction step of a sample occurs (e.g., where solutionincludes an extraction buffer). Furthermore, the lower chamber cancomprise a filter through which an extracted sample flows. For example,if a filter is disposed at the proximal end of—the lower chamber, anextracted sample then flows through a filter (e.g., a mesh disk) therebyprecluding certain components of the extraction mixture from passinginto the reagent area compartment comprising a solid reagent bead.Furthermore, a filter means can also function to restrain the reagentbead during SCD transportation and storage. As noted herein, the reagentbead can comprise both the detection and capture probe, or two separatebeads can each contain detection or capture probes.

The filtering aspect allows an analyte of interest to migrate throughthe device in a controlled fashion with few, if any, interferingsubstances. The filtering aspect, if present, often provides for a testhaving a higher probability of success, depending on the type of samplebeing processed, as would be evident to one of skill in the art (e.g.,whole blood sample versus throat swab). In another embodiment, the SCDmay also incorporate reagents useful to avoid cross-reactivity withnon-target analytes that may exist in a sample and/or to condition thesample; depending on the particular embodiment, these reagents mayinclude, but not limited to, non-hCG blockers, anti-RHO reagents,Tris-based buffers, EDTA, among others. When the use of whole blood iscontemplated, anti-RBC reagents are frequently utilized. In yet anotherembodiment, the SCD may incorporate other reagents such as ancillaryspecific binding members, fluid sample pretreatment reagents, and signalproducing reagents (e.g., substrates necessary for reacting with labelconjugates).

In another embodiment, the lower chamber can comprise a small element ofabsorbent paper, on which a predetermined percentage of the extractedsample is retained for archival purposes. After passing through thecollection device and having a portin restrained for archivalpurposes,the extracted sample contacts a reagent solution or solid(e.g., conjugate bead 202), and the next assay step takes place as thespecimen liquid rapidly dissolves the conjugate bead and allows thereactants to mix and start the assay.

In other embodiments, the leer 105 disposed on the distal end of the SCDcan be replaced with or further attached to a valve structure that alsofunctions to control the flow of liquid out from the sample collectiondevice. A valve can also be utilized in practice of the invention, wherethe valve is disposed in the upper sealed chamber, thus providing ameans to control release of solutions contained therein. Whether in theupper chamber, lower chamber, or between any compartments disposed inthe SCD, a valve can be of any type as recognized in the art such as,but not limited to, a rotary, snap, breakable, stopcock, gate, ball,needle, butterfly, pinch, bellows, piston, slide, plug, diverter, oractuator valve. In one embodiment, the valve is a snap valve which isrendered open where pressure is exerted in the stem portion of the valvethat extends into the upper sealed chamber (100), thereby breaking openthe hollow stem 102 and allowing the contents of the sealed upperchamber to flow into the hollow tube, at the end of which is disposed asampling implement (e.g., swab 107).

In one embodiment, there is a valve at the distal end of the SCD. Whenthe valve is in the closed position, a sample or sample and reagent canbe retained in the lower chamber 104. When the valve is in the openposition the contents of a mixing subchamber—can be released, forexample by gravity flow. Alternatively, the bulb component of the samplecollection device is utilized to release the sample/reagent from theluer, luer-valve or valve. In a preferred embodiment of the presentinvention, the content from the distal or outlet end of the samplecollection device (“SCD”) is released from the SCD such that the flowcan be actuated, regulated or modulated.

In another embodiment, the distal end of the SCD is open, whereby priorto release of a solution from the upper sealed chamber, the SCD isengaged (e.g., by friction fit) into the receiving port of a TestDevice. In such an embodiment, the fluid flow from the distal end of theSCD into the Test Device is not regulated by a luer or a valvestructure.

In another embodiment, the distal end of the SCD does not utilize avalve but rather is open. The SCD may be attached to the test deviceprior to release of the buffer from the upper chamber. Upon release ofthe solution from the upper chamber, the sample is released and/orextracted from the collection implement by the solution and mixed withthe reagents located in the lower chamber. The mixture then flows to thetest device for analysis of the presence of one or more analytes. It ispossible to include water-dissolvable membranes within the lower chamberto slow the flow of the mixture out of the SCD onto the test device.Such membranes are conventional and can be designed to permit theretention of the mixture for differing periods of time sufficient toallow mixing and reaction of the reagents and sample analytes. Forexample, such membranes can be prepared from proteins, polysaccharidesor film formers.

In another embodiment, the distal end of an SCD comprises a very narrowopening that prevents fluid flow unless and until pressure is applied tothe device (e.g., via the bulb structure of the SCD, or if the housingis depressible, then by exerting pressure on the housing itself) toforce the fluid out from the distal end. In other words, there is novalve of any sort disposed at the distal end of the SCD.

In another aspect of the present invention the fluid regulatory means(e.g., luer 105, luer-valve combination and valve) is closed such thatthe sample or sample and one or more reagents can be retained in thelower chamber 104 for any length of time. The valve structure can thenbe mechanically, fully or partially, opened to release the contentsthrough the distal or outlet end of the SCD into a test device,optionally at a regulated or modulated rate.

Reagents utilized in an SCD of the invention can include one or moresalts, chelators, anticoagulants, detergents, stabilizers, diluents,buffering agents, enzymes, cofactors, specific binding members, labels,mucolytic and the like. The one or more reagents can be compounds thatfacilitate analysis of a sample. Furthermore, such reagents can readilybe adapted for use in a Test Device of the invention.

In a one embodiment the SCD is engaged to a second device, for examplethe test device of the present invention, such that opening of the valvestructure or removing a cap 106 covering the opening (no valve) releasesthe contents into the second device. In another embodiment, engaging theSCD comprising a male luer to the test device comprising a female luerresults in release of the contents of the reaction subchamber. In afurther embodiment, the contents from the distal end can be released viathe bulb component. Where a valve structure is utilized in the SCD, thevalve structure can be opened to release the contents by various meanssuch as, but not limited to, opening a stopcock or by turning, rotating,twisting or sliding the valve structure such that the valve can beopened to allow fluid communication into the test platform or byremoving a cap which opens the fluid flow path.

Archive Sample. In one aspect of the invention a means for archiving aportion of a sample is provided. In some embodiments, an SCD or TestDevice, or both, comprise an archival means, which can compriseanabsorbent or adsorbent substrate (e.g., paper or membrane),—a shortcapillary tube of defined length,or a small reservoir/compartment forretaining a portion of the sample in the lower chamber. In someembodiments, this archival mechanism is located at a position in thedevice before the sample encounters the reaction reagents.

In one embodiment shown in FIG. 28, a small compartment, that canprovide a small reservoir for an archive sample, 1109 is positioned inthe test device adjacent to the port/aperture 1108 for delivery ofsample to the test device. Such an archive compartment can be configuredto be removable or so that a substrate onto which the archive sample isdisposed, is itself removable from said compartment. For example, afilter/membrane material sized to fit into the compartment will functionto collect to a predetermined capacity of sample (e.g., cell, cellcomponents, protein, nucleic acid, etc.). The test device may contain apouch 1106 of wash buffer with a housing or cover 1107

In another embodiment, an SCD comprises a means for retaining an archivesample FIG. 29A. For example, within a SCD lower compartment, filterpaper and hydrophobic membranes can be provided configured to retain asample for archiving purposes. Various combinations of materials arepossible. In one embodiment the means for archiving comprises threedisks 1203, 1206, 1207, that may or may not touch each other. In anotherembodiment, the SCD FIG. 29A can be configured with sleeves 1202 whichprovide a means to move the sizes of the tube/casing closer to the swab1205 attached to stem 1201 so that as a fluid exits the swab it willstay in close proximity to the swab, so as to improve the efficiency ofextracting fluid from the swab. A reagent bead is depicted as 1208 andthe exit port is 1204.

As shown in FIG. 29B the disks can comprise a grid portion 1210 and apad portion 1209, wherein the pad portion is designed to retain anarchive sample. The pad portion can be comprised of anyabsorptive/adsorptive material and can comprise 5, 10, 15, 20, 25, 30,35, 40, 45 or 50% of the surface area of a disk. Furthermore, the gridportion can comprise three dimensional (“3D”) substrates raised relativeto the surface of a disk. Such 3D protrusions can provide a grid intowhich a reagent bead can be disposed. Such beads can measure in sizefrom about 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5.0, 5.5, to about6.0 nm.

In one embodiment, the archived material is a cell(s) or cellularcomponent, including but not limited to a protein, peptide, proteinfragment or nucleic acid molecule. Therefore, samples can be preservedfor further testing depending on the type of molecule archived (e.g.,protein versus nucleic acid). Furthermore, archive disks 1207(forexample as shown in FIG. 29A) provide a means of storing samples andmaintain stability of said samples from about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 21 to 30 days. For example, RNA is stable for about2, 3, 4, or 5 days.

In another embodiment, the archival disks are placed in a preservativesolution, which extends storage time for said archive samples from about1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks. Of course depending on thein-field setting, samples can be stored indefinitely (e.g., once thesample is subjected to freezing).

In another embodiment, a reaction compartment—in the lower chamber canbe removed from the sample receiving tube and placed in a housing (e.g.,plastic tube). In one embodiment, the compartment (e.g., a cage) retainsa small volume of sample mixture to which a preservative can be addedfor storage. In another embodiment, the solutions provided in the upperchamber or a reaction solution in the lower chamber can also includepreservatives necessary to archive a liquid sample. Such preservativesare known in the art. See, e.g., U.S. Pat. RE29061; Buccholz et atTransfusion. 1999 September; 39(9):998-1004; Quiagen specialty reagents,available at Quiagen.com.

In one embodiment, an archive sample is retained for later testing(e.g., by RT-PCR, See Example 8).

Sample identification. The SCD also includes anywhere on the samplingimplement or the sample receiving tube, for example, attached to theouter tube 103, 3 identical identifying labels (e.g., barcodes allowingat least 10⁹ unique values) for patient identification number. Thelabels can be peel-off and is self-adhesive. One label is retained onthe SCD 103. The peel-off copies can be placed on the Test Device FIG. 3and on any facility paperwork, or an archival reservoir means. Bar codeformat will be to a universal standard such as Codabar 303 or 305. Inother embodiments, the identifying labels can be signal emittingtransponders known in the art, including but not limited to, radiofrequency emitter, light emitter or electromagnetic wave emitter.

Compartments in the Upper or Lower Chambers. In some aspects of theinvention, the SCD comprises one or more compartments in the lowerchamber that can include reagents, filters, membranes and reservoirs. Inone embodiment the upper chamber of the SCD may comprise one, two ormore compartments-, each of which can further contain a solution. Insome embodiments, such compartments can comprise the same or twodifferent solutions, reagents, buffers, or a combination thereof.Further, multiple compartments can be arranged in series in a lowerchamber (e.g., multiple cages—in series). In addition, such compartmentsmay be referred to as “subcompartment” or “subcompartments” in thedisclosures herein.

In one embodiment, compartment is distal relative to a samplingimplement and comprises a liquid or solid reagent component thatcomprises binding agents that are specific to a particular analyte (oranalyte type). For example, the liquid or solid reagent componentincludes a specific binding agent (e.g., antibody) that is capable ofspecifically binding an analyte that may be present in a sample. In someembodiments, a single reaction or mixing compartment—is utilized in theSCD that is distal to and in fluid communication with the samplingimplement. In other embodiments, one or more compartments can beutilized where one compartment functions as a lysis or extractionchamber, while a second compartment distal to the first compartmentfunctions as a reagent-sample mixing chamber. In further embodiments,-filtering means may be disposed on the proximal end of one or morecompartments, which compartment(s) is disposed—distal relative to thesampling implements. Filter means can be utilized to remove certaincomponents from the sample, prior to extraction/lysis, sample-reagentmixing, during processing or before release from the SCD. Furthermore,the same or different filtering means can be disposed on multiplecompartments if such multiple compartments are present in the samplereceiving tube.

Samples.

A sample is any material to be tested for the presence and/orconcentration of an analyte. In general, a biological sample can be anysample taken from a subject, e.g., non-human animal or human andutilized in the test devices. For example, a biological sample can be asample of any body fluid, cells, or tissue samples from a biopsy. Bodyfluid samples can include without any limitation blood, urine, sputum,semen, feces, saliva, bile, cerebral fluid, nasal swab, urogenital swab,nasal aspirate, spinal fluid, etc. Biological samples can also includeany sample derived from a sample taken directly from a subject, e.g.,human. For example, a biological sample can be the plasma fraction of ablood sample, serum, protein or nucleic acid extraction of the collectedcells or tissues or from a specimen that has been treated in a way toimprove the detectability of the specimen, for example, a lysis buffercontaining a mucolytic agent that breaks down the mucens in a nasalspecimen significantly reducing the viscosity of the specimen and adetergent to lyse the virus thereby releasing antigens and making themavailable for detection by the assay. A sample can be from any subjectanimal, including but not limited to, human, bird, porcine, equine,bovine, murine, cat, dog or sheep.

For example, a sample can be derived from any source, such as aphysiological fluid, including blood, serum, plasma, saliva or oralfluid, sputum, ocular lens fluid, nasal fluid, nasopharyngeal or nasalpharyngeal swab or aspirate, sweat, urine, milk, ascites fluid, mucous,synovial fluid, peritoneal fluid, transdermal exudates, pharyngealexudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinalfluid, semen, cervical mucus, vaginal or urethral secretions, amnioticfluid, and the like. Herein, fluid homogenates of cellular tissues suchas, for example, hair, skin and nail scrapings and meat extracts arealso considered biological fluids. Pretreatment may involve preparingplasma from blood, diluting or treating viscous fluids, and the like.Methods of treatment can involve filtration, distillation, separation,concentration, inactivation of interfering components, and the additionof reagents. Besides physiological fluids, other samples can be usedsuch as water, food products, soil extracts, and the like for theperformance of industrial, environmental, or food production assays aswell as diagnostic assays. In addition, a solid material suspected ofcontaining the analyte can be used as the test sample once it ismodified to form a liquid medium or to release the analyte. Theselection and pretreatment of biological, industrial, and environmentalsamples prior to testing is well known in the art and need not bedescribed further.

Other fields of interest include the diagnosis of veterinary diseases,analysis of meat, poultry, fish for bacterial contamination, inspectionof food plants, restaurants, hospitals and other public facilities,analysis of environmental samples including water for beach, ocean,lakes or swimming pool contamination. Analytes detected by these testsinclude viral and bacterial antigens as well as chemicals including, forexample, lead, pesticides, hormones, drugs and their metabolites,hydrocarbons and all kinds of organic or inorganic compounds.

Test Device.

The present disclosure provides a test device, particularly immunoassaydevices, for determining the presence or absence of multiple analytes ina fluid sample. In general, a Test Device FIG. 3 of the presentdisclosure includes a matrix defining an axial flow path. Typically, thematrix further includes a sample receiving zone, one or more test zonesand one or more control zones. In frequent embodiments, a test regioncomprises the test and control zones, which are collectively addressablelines.

As used herein in the context of the Test Device the terms “axial flowmembrane”, “lateral flow membrane”, “test membrane”, “test strip” or“matrix” are used interchangeably which employs capillary action to moveor transport the test fluids or employs the movement of fluid separatefrom capillary action as where fluid is pumped by the accumulation ofgas pressure, hydraulic pressure (direct pumping using a piston orrotary, bellows or other type pump on the assay fluids, electrostaticmovement due to an electric field, gravity, etc.).

In one aspect of the invention, the Test Device as depicted in FIG. 3 iscomprised of an aperture/port into which the distal end of a SCD of theinvention is engaged either by friction fit, luer lock, adaptor orvalve. An aperture/port 302,—provides an opening through which a samplefrom the SCD flows into the Test Device. A blood separation membrane canbe disposed at the port which provides one way flow. In anotherembodiment, such a membrane can also be disposed in the SCD (e.g.,immediately distal to the sample swab implement.

Upstream of the aperture is a compartment 307 that may be in fluidcommunication with the aperture, which aperture is in fluidcommunication with a wicking substrate 309. Furthermore, the compartmentcan comprise one or more subcompartments 308 that contain one or moresolution(s). Subcompartments in the context of the Test Device can bemade of a pierceable, puncturable, breakable (e.g., ampule) ordepressible bladder like material. In one embodiment, the collar breaksaway, however, the buffer is only released once additional force isapplied to the buffer/wash compartment 308. As indicated herein, suchcompartments can be manipulated by applying pressure so as to puncture,break or depress the compartment enough so to release it contents. Inaddition, such compartments may be pierced by a lance, stab or appendagethat breaks into said compartment upon exertion of force (e.g., thumbpressing down) onto said compartment.

In one embodiment, the Test Device comprises two sections, wherein onesection comprises a portion where a sample is applied 302—and acompartment upstream of portion where the sample is applied comprising awash or running buffer 308. In another embodiment, the upstream sectioncan comprise one or more (e.g., two) compartments which may contain thesame or different buffers, wherein each compartment can be separately orsimultaneously manipulated to expel its contents.

In another embodiment, the compartment 307 itself can be semi-rigid,pliable or depressible, or bladder like, which provides a means forcompacting the compartment to expel any contents therein. Therefore, insome embodiments, a user can exert pressure on the compartment 307 thatwill result in contents therein, whether self-contained or contained ina subcompartment, to be released to the wicking substrate 309. Such asolution can function as a wash or chase buffer, mobilizing or enhancingmobilization of the processed sample mixture through the wicking pad andinto the test strip 310. Generally, such liquid solutions can comprisewash buffer, saline or any desired solution. Furthermore, in someembodiments, such a solution can comprise reagents, enzymes, labels orchemical compounds. The wash buffer mobilizes any unbound label causingit to migrate along the strip past the detection zone thus reducingbackground.

Furthermore, downstream of the test strip 310 is disposed an absorbentsubstrate 311. The test membrane substrate can overlap or abut to one orboth the wicking substrate and absorptive substrate, respectively.Furthermore, in some embodiments, the Test Device upper 306 or lowerhousing 312 can comprise identity labels 303 and 305, which identify andcorrespond to an identical identity label on the SCD and can alsoidentify the lot number of the Test Device (e.g., for quality assuranceand tracking purposes). Window 304 through the upper housing permitsvisualization and reading of the results.

In a related embodiment, the matrix further includes an absorbent zonedisposed downstream of the lateral flow substrate, membrane or matrix.In one embodiment, a wicking pad can be disposed upstream of the lateralflow membrane. In another embodiment, a wicking pad is disposed directlybelow the sample entry aperture. Moreover, in preferred embodiments, thetest region, which comprises the test and control zones, is observableFIG. 8.

Suitable materials for manufacturing absorbent substrates 311 include,but are not limited to, hydrophilic polyethylene materials or pads,acrylic fiber, glass fiber, filter paper or pads, desiccated paper,paper pulp, fabric, and the like. For example, the lateral flow membraneabsorbent zone may be comprised of a material such as a nonwovenspunlaced acrylic fiber, i.e., New Merge (available from DuPont) or HDKmaterial (available from HDK Industries, Inc.), nonwoven polyethylenetreated to improve the hydrophobic property of the material.

Safety Means. In some embodiments, a safety means 301 is disposed overthe depressible chamber—307 so that the contents of the chamber cannotbe accidentally discharged into the channel in fluid communication withthe lateral flow membrane. A safety means can be a cover or flange thatis lifted or pulled back to expose the depressible chamber or a pushbutton disposed thereon.

Furthermore, such a safety means can function as an adaptor for aspecific cognate adaptor, luer or valve present on the distal end of theSCD. Thus, a safety means can cover an aperture into which the distalend of the SCD is engaged, for example, prior to release of a sampleinto the Test Device. In an additional embodiment, the reader isdesigned so that a Test Device can only be inserted into a receivingport if the safety cover is first removed. For example, a Test Devicewith its safety cover removed indicates that a sample has beenintroduced into the Test Device and running buffer has been releasedfrom the compartment 307 upstream of the aperture (adapter/safetycover). In one embodiment, the aperture is disposed above the wickingpad 309.

Gap Means. In some embodiments, a Test Device comprises a gap disposedbetween the lateral flow membrane (e.g., wicking pad) and the channel influid communication with the buffer reservoir. The gap functions to keepany solution contained in the push button reservoir and assay sampleseparate until the appropriate time according to the assay developmentFIG. 7. In some embodiments, the gap can be from about 0.5, 1, 1.5, 2,2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 mm. In one embodiment the gap isgreater than zero and less than 3 mm. Thus, where a user exerts pressureon the compartment—upstream of the sample aperture-, the gap as shownbetween 703 and 704 is forced closed and a solution contained in thecompartment—flows in the direction shown by 701 to and through thewicking pad, thus mobilizing the sample through the test strip-. Asindicated above, the solution can comprise any desired buffer, reagent,chemical compound, dye, label or bead. It should be understood that thegap embodiments disclosed herein can be adapted to any of the TestDevice configurations disclosed herein.

In one embodiment, the test device is a lateral flow test strip,preferably, though not necessarily, encased in a housing, designed to beread by the reader FIGS. 3, 27.

In one embodiment, shown in FIGS. 27 A, B, and C a SCD-processed sampleis introduced into the Test Device 1001, a chase or running buffer—issubsequently released and follows the specimen through the wicking padand into the test strip—, 1003 where specifically patterned captureagents bind their partner capture probes. Therefore, if a particularanalyte is present, it will be bound by a detection probe and captureprobe (as described herein above), which capture probe will bind itsspecific partner capture moiety immobilized on defined spots or lines onthe test strip—1003. FIG. 27B shows the components of the test deviceincluding the upper housing 1002, lower housing 1004, and aperture 1005.

In one embodiment, a wash/running buffer solution is comprised in afoil, sac or buster type packet (e.g., similar to ketchup/condimentpacket) which is disposed in the Test Device upstream FIG. 27C of thesample entry port 302, 504, 605, 1005, 1108. The sac or packet can bedesigned so that it is symmetric about the two orthogonal axes so thatit can be loaded into the Test Device easily. Therefore, in oneembodiment, the cover of the Test Device disposed over the packet whenpressed down can cause the packet to break releasing the contentstherein.

In another embodiment, the button portion can comprise a piercingappendage that punctures the packet as the button is depressed thusreleasing the contents therein. A leaf spring or cantilever spring FIG.27C can rest between the packet and the button and results in pressureexerted on the packet to ensure all the contents are released. Further,the geometry of the Test Device is configured so that the wash buffer isdirected toward the wicking pad 904/905, 1006. In addition the geometryof the button, spring, and housing also reduces air voids in the packetarea allowing the wash buffer to flow in any direction, even againstgravity (e.g., uphill), as necessary, but not back into the packetstorage area.

The number and size of the holes created, as well as the geometry of thehole created can be adjusted relative to one another in order to allowfor predetermined flow of the wash buffer out of the packet.

In one embodiment, the piercing appendage (e.g., needle) will provide afluid resistance barrier on the top of the packet, allowing fluid toexit the lower portion of the packet in the direction of the wickingpad. The piercing needle can also be tapered in order to achieve orenhance this function.

In one embodiment, the spring FIG. 27C is an integral part of thebutton, top housing or lower housing FIG. 27B or it can be a separatecomponent altogether that is configured to easily fit and seal thewash/running buffer chamber.

In one embodiment, the sides of the button are designed to minimizepinch points while the button is depressed. Sides can also be designedto provide a baffle-type function, minimizing the risk of liquid exitingthe Test Device.

In another embodiment, the geometry of the feature that supports the endof the wicking strip is designed to allow the piercing feature (e.g.,needle) to pass through the packet and not allow the packet to form aseal between the packet and the support feature. The action of theneedle pierces both the wicking pad and the packet. In anotherembodiment, the piercing is only of the packet with the wicking padlocated directly adjacent to the pierced hole.

In one embodiment, the wash/running buffer in the Test Device iscomprised in a breakable/rupturing substrate (e.g., an ampoule).Pressure exerted on a sealing membrane or button breaks the ampoule thusreleasing its contents. In one embodiment, a channel, gutter, or troughis designed to direct the buffer to the wicking pad.

In one embodiment, the aperture for receiving the SCD distal endcomprises a break-away collar (“Lock Collar”) 1104 (FIG. 28A)which—attaches to the SCD assembly and breaks away from the Test Devicebody 1105 as the SCD 1101 is removed, thus releasing wash or runningbuffer from a compartment/reservoir 1102 upstream or immediatelyupstream of said aperture. In yet another embodiment, the Lock Collarwhen twisted into the lock position allows a sample to be dispensed ontothe Test Device while concurrently releasing buffer or wash buffer froman upstream compartment. For example, the Lock Collar will comprise ageometry of channels 1103, holes or openings that line up with openings,channels or holes of the wash/buffer compartment only when the collar isin the lock position. Such a Lock Collar can be utilized with any of theone or more upstream compartments 1102, 1106 that can be utilized todeliver a buffer/wash or any other liquid.

In an alternative embodiment, the SCD 1101 can comprise the Lock Collar1103 which fits into the Test Device body 1103 and twists from an unlockposition to a lock position 1104.

In one embodiment as depicted in FIG. 28B, the upstream wash/buffercompartment comprises a soft membrane (e.g., form fill seal pack) orampoule that is easily ruptured/broken upon exertion of minimal force(e.g., user pressing with finger). Such an onion skin compartment—1106,can be further covered by a hard removable cover 1107 which preventsaccidental breakage of the onion skin. The sample enters the test devicethrough a port 1109 and the device may have a narrow channel 1109 forrecovery of an archival sample.

As shown in FIG. 8 the sample is delivered to the test strip by the SCD802 which includes the stem and swab 806. Upstream of the test strip isthe compartment 801 with wash buffer or other fluid. The test stripincludes test zones 803, and control zones A, B, C 804. The detectionprobe, via the conjugate label, will provide a detectable signal. TheTest Device is then inserted into a reader 400 as shown in FIG. 4, wherethe signal from the label is measured and/or detected. In anotherembodiment, the test strip 401 can he inserted into a moveable tray 402in the reader after the short assay processing period has completed fora very short read period (˜20 seconds), this allows for a much higherthrough put of tests with one reader. Further, in another embodiment,the test strip can be inserted into the reader prior to addition of thesample.

In any of the embodiments herein directed to a wash/running bufferrelease from a chamber upstream of the sample (e.g., sample entry port),a time delay feature can be configured into the Test Device, so that aperiod of time passes between introduction of the sample and the releaseof the wash/running buffer. For example, a dry wicking pad substrateswells when wet (i.e., after wash buffer release) and due to theswelling connects and otherwise disconnected wicking strip FIG. 34. Forexample, a sample is applied 1405. The ampoule or substrate 1401comprising the wash buffer is broken/ruptured to release the liquid intothe dry wicking pad portion 1402, which swells 1403 and provides liquidcommunication to the wicking pad portion containing the sample 1405. Thesample/buffer run through the test strip via the wicking pad 1406.

In another embodiment, a predetermined length/density of fibrousmembrane is placed in between the wash buffer compartment and thewicking membrane, which fibrous membrane can delay the contact of thewash buffer to the wicking membrane thus functioning as a time delaymechanism. Buffer wicks down the fibrous membrane and accumulates on theend of the membrane fibers until it reaches the wicking membrane andflows through with the sample disposed on the wicking membrane. Inanother embodiment, the buffer accumulates at the ends of the membranefibers until there is enough volume to bridge a gap separating thefibrous membrane from the wicking membrane.

In other embodiments, a plunger or spring mechanism is configured intothe Test Device, which functions to reduce the compartment/ampoulevolume, thus ensuring the contents therein are dispersed onto a wickingpad. A plunger can be moved forward by the user exerting pressure on thebutton or a spring loaded plunger can be driven forwarded in anautomated fashion (e.g., when placed in the reader). The plunger forms aseal as it drives forward so that the liquid's only means of exit isthrough to the wicking pad.

In one embodiment, the liquid transport along the test strip is basedupon capillary action. In a further embodiment, the liquid transportalong the matrix is based on non-bibulous lateral flow, wherein all ofthe dissolved or dispersed components of the liquid sample are carriedat substantially equal rates and with relatively unimpaired flowlaterally through the matrix, as opposed to preferential retention ofone or more components as would occur, e.g., in materials that interact,chemically, physically, ionically or otherwise with one or morecomponents. See for example, U.S. Pat. No. 4,943,522, herebyincorporated by reference in its entirety.

Any suitable material can be used to make the devices disclosed herein,such material including a rigid or semi-rigid, non-water-permeablematerial, such as glass, ceramics, metals, plastics, polymers, orcopolymers, or any combination thereof. In some embodiments, either theSCD or Test Device comprise a plastic, polymer or copolymer such asthose that are resistant to breakage, such as polypropylene,polyallomer, polycarbonate or cycloolefins or cycloolefin copolymers.Furthermore, devices of the invention can be made by appropriatemanufacturing methods, such as, but not limited to, injection molding,blow molding, machining or press molding.

As used herein, test strip substrate refers to the material to which acapture moiety is linked using conventional methods in the art. Avariety of materials can be used as the substrate, including anymaterial that can act as a support for attachment of the molecules ofinterest. Such materials are known to those of skill in this art andinclude, but are not limited to, organic or inorganic polymers, naturaland synthetic polymers, including, but not limited to, agarose,cellulose, nitrocellulose, cellulose acetate, other cellulosederivatives, dextran, dextran-derivatives and dextran co-polymers, otherpolysaccharides, glass, silica gels, gelatin, polyvinyl pyrrolidone(PVP), rayon, nylon, polyethylene, polypropylene, polybutlyene,polycarbonate, polyesters, polyamides, vinyl polymers,polyvinylalcohols, polystyrene and polystyrene copolymers, polystyrenecross-linked with divinylbenzene or the like, acrylic resins, acrylatesand acrylic acids, acrylamides, polyacrylamide, polyacrylamide blends,co-polymers of vinyl and acrylamide, methacrylates, methacrylatederivatives and co-polymers, other polymers and co-polymers with variousfunctional groups, latex, butyl rubber and other synthetic rubbers,silicon, glass, paper, natural sponges, insoluble protein, surfactants,red blood cells, metals, metalloids, magnetic materials, or othercommercially available media or a complex material composed of a solidor semi-solid substrate coated with materials that improve thehydrophilic property of the strip substrate, for example, polystyrene,Mylar, polyethylene, polycarbonate, polypropylene, polybutlyene, metalssuch as aluminum, copper, tin or mixtures of metals coated with dextran,detergents, salts, PVP and/or treated with electrostatic or plasmadischarge to add charge to the surface thus imparting a hydrophilicproperty to the surface.

In one embodiment, the lateral flow membrane is comprised of a porousmaterial such as high density polyethylene sheet material manufacturedby Porex Technologies Corp. of Fairborn, Ga., USA, The sheet materialhas an open pore structure with a typical density, at 40% void volume,of 0.57 gm/cc and an average pore diameter of 1 to 250 micrometers, theaverage generally being from 3 to 100 micrometers. In anotherembodiment, the label zone is comprised of a porous material such as anonwoven spunlaced acrylic fiber (similar to the sample receiving zone),e.g., New Merge or HDK material. Often, the porous material may bebacked by, or laminated upon, a generally water impervious layer, e.g.,Mylar. When employed, the backing is generally fastened to the matrix byan adhesive (e.g., 3M 444 double-sided adhesive tape). Typically, awater impervious backing is used for membranes of low thickness. A widevariety of polymers may be used provided that they do not bindnonspecifically to the assay components and do not interfere with flowof the fluid sample. Illustrative polymers include polyethylene,polypropylene, polystyrene and the like. On occasion, the matrix may beself-supporting. Other membranes amenable to non-bibulous flow, such aspolyvinyl chloride, polyvinyl acetate, copolymers of vinyl acetate andvinyl chloride, polyamide, polycarbonate, polystyrene, and the like, canalso be used. In yet another embodiment, the lateral flow membrane iscomprised of a material such as untreated paper, cellulose blends,nitrocellulose, polyester, an acrylonitrile copolymer, and the like. Thelabel zone may be constructed to provide either bibulous or non-bibulousflow, frequently the flow type is similar or identical to that providedin at least a portion of the sample receiving zone. In a frequentembodiment, the label zone is comprised of a nonwoven fabric such asRayon or glass fiber. Other label zone materials suitable for use by thepresent invention include those chromatographic materials disclosed inU.S. Pat. No. 5,075,078, which is herein incorporated by reference.

In a frequent embodiment, the test strip substrate is treated with asolution that includes material-blocking and label-stabilizing agents.Blocking agents include bovine serum albumin (BSA), methylated BSA,casein, acid or base hydrolyzed casein, nonfat dry milk, fish gelatin,or similar. Stabilizing agents are readily available and well known inthe art, and may be used, for example, to stabilize labeled reagents. Insome embodiments, the upstream compartment containing a solution 307 cancomprise multiple ampules, which can be selectively punctured or brokento release their contents. Therefore, in one embodiment, blockingreagents are contained in one ampule which is utilized to pre-treat(e.g., “block”) the test strip (i.e., lateral flow membrane), while theadditional ampule is reserved for washing the sample through the teststrip.

In various disclosures herein, the test strip/lateral flow membranecomprises multiple test zones, one of which is referenced 803 in FIG. 8.Test zones generally contains a pre-selected capture moieties, where apre-selected region comprises capture moieties that are partners forcapture moieties conjugate to analyte-specific binding agents, such asmonoclonal antibodies. In many of the presently contemplatedembodiments, multiple types of labeled reagents are incorporated in SCDsuch that they may permeate together with a fluid sample contacted inthe device. These multiple types of labeled reagent can be analytespecific or control reagents and may have different detectablecharacteristics (e,g., different colors) such that one labeled reagentcan be differentiated from another labeled reagent if utilized in thesame device, or in a preferred embodiment, having different capturemoieties. As the labeled reagents are frequently bound to a specificanalyte of interest and are subsequently processed through to a TestDevice comprising a test strip, differential detection of labeledreagents having different specificities (including analyte specific andcontrol labeled reagents) may be a desirable attribute. However,frequently, the ability to differentially detect the labeled reagentshaving different specificities based on the label component alone is notnecessarily due to the presence of defined test and control zones in thedevice, which allow for the accumulation of labeled reagent indesignated zones.

In some embodiments, each detection probe is conjugated to a fluorescentlabel emitting a different wavelength. Therefore, where a plurality ofspecific binding agents are comprised in a SCD, where the pluralitycomprises several different groups of specific binding pairs, eachbinding pair for a given group comprises labels different than any othergroup, where the multiple groups make up the plurality of specificbinding agents. For example, a group of specific binding antibodies toinfluenza A can be conjugated to one type of fluorescent label (i.e.,detection probes conjugated to a first fluorescent label), while a groupof specific binding antibodies (i.e., detection probes conjugated to asecond fluorescent label), and a third, fourth or fifth group can eachcomprise detection probes conjugated to different fluorescent labels. Ofcourse, it should be evident that detection probes can also comprise acombination of different types of labels (e.g., first group comprising afluorescent label, while second group a metal, while a control cancomprise a chromophore). In one embodiment, the fluorescent labels emitwavelengths that are sufficiently distinct so that several test linescan be differentiated.

The present description provides for the development and use of singleor multiple control zones in a single immunoassay device that arepositioned in a predetermined manner relative to individual test zonesthereby allowing easy identification of each of the one or more analytesof interest tested for in the device. The present description furtherprovides for the making of control zones of various shapes, physical orchemical identities, and colors. In part, the use of such control zonesallows for immunoassay devices that are easy to use, and allow for theidentification of multiple analytes during a single assay procedure.(See, Example 11).

In one embodiment, the Test Device does not include any reagentscontained therein that are capable of specifically binding to an analyte(e.g., antibody that is specific for H5N1).

The test region generally includes one or more control zone that isuseful to verify that the sample flow is as expected. Each of thecontrol zones comprise a spatially distinct region that often includesan immobilized member of a specific binding pair which reacts with alabeled control reagent. In an occasional embodiment, the proceduralcontrol zone contains an authentic sample of the analyte of interest, ora fragment thereof. In this embodiment, one type of labeled reagent canbe utilized, wherein fluid sample transports the labeled reagent to thetest and control zones; and the labeled reagent not bound to an analyteof interest will then bind to the authentic sample of the analyte ofinterest positioned in the control zone. In another embodiment, thecontrol line contains antibody that is specific for, or otherwiseprovides for the immobilization of, the labeled reagent. In operation, alabeled reagent is restrained in each of the one or more control zones,even when any or all the analytes of interest are absent from the testsample.

In some embodiments, a labeled control reagent is introduced into thefluid sample flow either in the SCD or in the Test Device. For example,in the Test Device, control reagents can be included in the upstreamsolution/buffer reservoir, which are described herein FIG. 3. In anotherexample, the labeled control reagent may be added to the fluid samplebefore the sample is applied to the Test Device, e.g., present in themixing subchamber in the SCD.

Exemplary functions of the labeled control reagents and zones include,for example, the confirmation that the liquid flow of the sampleeffectively solubilized and mobilized the labeled reagents from the SCD,which are captured in one or more defined test zones. Furthermore,controls can confirm that a sufficient amount of liquid traveledcorrectly through the test strip test and control zones, such that asufficient amount of capture moieties could react with the correspondingspecific capture probes complexed to a specific analyte (i.e., via theantigen specific binding agent). Further, control reagents confirm thatthe immunocomplexes (e.g., analyte-analyte specific binding agent)migrate onto the test region comprising the test and control zones,cross the test zone(s) in an amount such that the accumulation of thelabeled analyte would produce a visible or otherwise readable signal inthe case of a positive test result in the test zone(s). Moreover, anadditional function of the control zones may be to act as referencezones which allow the user to identify the test results which aredisplayed as readable zones.

Since the devices of the present invention may incorporate one or morecontrol zones, the labeled control reagent and their correspondingcontrol zones are preferably developed such that each control zone willbecome visible with a desired intensity for all control zones afterfluid sample is contacted with the device, regardless of the presence orabsence of one or more analytes of interest.

In one embodiment, a single labeled control reagent will be captured byeach of the control zones on the test strip. Frequently, such a labeledcontrol reagent will be deposited onto or in the label zone in an amountexceeding the capacity of the total binding capacity of the combinedcontrol zones if multiple control zones are present. Accordingly, theamount of capture reagent specific for the control label can bedeposited in an amount that allows for the generation of desired signalintensity in the one or more control zones, and allows each of thecontrol zones to restrain a desired amount of labeled control-reagent.At the completion of an assay, each of the control zones preferablyprovide a desired and/or pre-designed signal (in intensity and form).Examples of contemplated pre-designed signals include signals of equalintensities in each control zone, or following a desired pattern ofincreasing, decreasing or other signal intensity in the control zones.

In another embodiment, each control zone will be specific for a uniquecontrol reagent. In this embodiment, the label zone may include multipleand different labeled control reagents, equaling the number of controlzones in the assay, or a related variation. Wherein each of the labeledcontrol reagents may become restrained in one or more pre-determined andspecific control zone(s). These labeled control reagents can provide thesame detectible signal (e.g., be of the same color) or providedistinguishable detectible signals (e.g., have different colored labelsor other detection systems) upon accumulation in the control zone(s).

In yet another embodiment, the control zones may include a combinationof the two types of control zones described in the two previousembodiments, specifically, one or more control zones are able torestrain or bind a single type of labeled control reagent, and othercontrol zones on the same test strip will be capable of binding one orseveral other specifically labeled control reagents.

In one embodiment, the labeled control reagent comprises a detectiblemoiety coupled to a member of a specific binding pair. Typically, alabeled control reagent is chosen to be different from the reagent thatis recognized by the means which are capable of restraining an analyteof interest in the test zone. Further, the labeled control reagent isgenerally not specific for the analyte. In a frequent embodiment, thelabeled control reagent is capable of binding the corresponding memberof a specific binding pair or control capture partner that isimmobilized on or in the control zone. Thus the labeled control reagentis directly restrained in the control zone.

In another embodiment, the detectable moiety which forms the labelcomponent of the labeled control reagent is the same detectible moietyas that which is utilized as the label component of the analyte ofinterest labeled test reagent. In a frequent embodiment, the labelcomponent of the labeled control reagent is different from the labelcomponent of the labeled test reagent, so that results of the assay areeasily determined. In another frequent embodiment, the control label andthe test label include colored beads, e.g., colored latex. Alsofrequently, the control and test latex beads comprise different colors.

In a further embodiment, the labeled control reagent includesstreptavidin, avidin or biotin and the control capture partner includesthe corresponding member of such specific binding pairs, which readilyand specifically bind with one another. In one example, the labeledcontrol reagent includes biotin, and the control capture partnerincludes streptavidin. The artisan will appreciate that other members ofspecific binding pairs can alternatively be used, including, forexample, antigen/antibody reactions unrelated to analyte. In yet otherembodiment, capture partners can include any of the binding moietiesdisclosed herein.

The use of a control zone is helpful in that appearance of a signal inthe control zone indicates the time at which the test result can beread, even for a negative result. Thus, when the expected signal appearsin the control line, the presence or absence of a signal in a test zonecan be noted.

In still further embodiment, a control zone comprising a mark thatbecomes visible in the test region when the test region is in a moiststate is utilized. Control zones of this type are described in U.S.patent application Ser. No. 09/950,366, filed, Sep. 10, 2001, currentlypending and published as U.S. patent application Publication No.20030049167, and Ser. No. 10/241,822, filed Sep. 10, 2002, currentlypending and published as U.S. patent application Publication No.20030157699.

In occasional embodiments, one or more control zones of this type areutilized. In another embodiment, a combination of control zones of thetype utilizing labeled control reagents and control zone and of the typethat display the control zone when in a moist state can be used. Thisallows a simple way to formulate control zones while allowing to use areagent-based control zone to ascertain that the re-solubilization andmobilization of the reagents in SCD-processed samples has beeneffective, and that the specific reactions took place as expected, allalong the path defined Test Device, wick, test strip and absorbent pad.The present embodiment includes the use of one or more control zonesthat become visible when the test region is in the moist state for eachof the control zones of an assay, except the control zone on the distalor downstream end of the test strip.

The present description further provides means to build a rapid,multi-analyte assay, which is needed in many fields of environmentalmonitoring, medicine, particularly in the field of infectious disease.For example, contemplated devices include those useful for thedifferential diagnosis of Flu A or Flu B, and subtypes thereof (e.g.,Flu A, H5N1) which may result in different treatments, or thedifferential diagnosis of Flu A, Flu B, and/or RSV in one step. Suchdevices permit the use of a single sample for assaying multiple analytesat once, and beneficially allows for a considerable reduction of thehands-on time and duration of the diagnostic process for the benefit ofthe doctor, or user in general. As such a plurality of immunoreagentscan be utilized in an SCD of the invention, where said pluralitycomprises populations of specific binding agents, comprising pairsconjugated respectively to label and capture moiety, whereby saidplurality comprise multiple populations each specific for a differentanalyte as compared to any other population. For example, the pluralityof immunoreagents can be specific for several types of one pathogen ordifferent pathogens.

A variety of analytes may be assayed utilizing devices and methods ofthe present disclosure. In a particular device useful for assaying forone or more analytes of interest in a sample, the collection of analytesof interest may be referred to as a panel. For example, a panel maycomprise any combination (or all of) of influenza A, influenza B,influenza A subtypes, respiratory syncytial virus (RSV), adenovirus, anddifferent types of Parainfluenza viruses (for example Types 1, 2, 3etc.). Another panel may comprise testing for a selection of one or moreof upper respiratory infection including, for example, Streptococcuspneumoniae, Mycoplasma pneumoniae and/or Chlamydia pneumoniae. Yetanother panel can be devised for the diagnosis of sexually transmitteddisease including, for example, Chlamydia, Trichomonas and/or Gonorrhea.In each case, a particular panel devised to provide signals on the TestDevice for a particular series of analytes is readily obtained byincorporating a different set of detection and capture probes in theSCD, which is described herein. Therefore, a particular SCD will provideall the reagents necessary to detect a particular panel of analyteswhich are detected when using a Test Device employing test strips thathave detection reagents that are not specific for the analytes ofinterest. In other embodiments, a broad scope Test Device can comprisenon-specific capture moieties for several series of analytes fromrelated or distinct pathogens, e.g., detection of HIV and HCV antigens;HIV and tuberculosis, Influenza A, B, and subtypes of A, bacterial andviral infections. Thus a single Test Device can be used with SCDscomprising immunoreagents for a different panel of analytes, providingenhanced efficiency and cost effectiveness.

For example, a panel may optionally include a variety of other analytesof interest, including SARS-associated coronavirus, influenza A; ahepatitis panel comprising a selection of hepatitis B surface Ag or Ab,hepatitis B core Ab, hepatitis A virus Ab, and hepatitis C virus; aphospholipids panel comprising a selection of Anticardiolipin Abs (IgG,IgA, and IgM Isotypes); an arthritis panel comprising a selection ofrheumatoid factor, antinuclear antibodies, and Uric Acid; an EpsteinBarr panel comprising a selection of Epstein Barr Nuclear Ag, EpsteinBarr Viral Capsid Ag, and Epstein Barr Virus, Early Antigen; otherpanels include HIV panels, Lupus panels, H. Pylori panels, toxoplasmapanels, herpes panels, Borrelia panels, rubella panels, cytomegaloviruspanels, panels testing for recent myocardial infarction with analytescomprising an isotype of Troponin with myoglobin and/or CKMB and manyothers. One of skill in the art would understand that a variety ofpanels may be assayed via the immunoassays utilizing the devicesdisclosed herein. Immunoassay methods are known in the art. See, e.g.,CURRENT PROTOCOLS IN IMMUNOLOGY (Coligan, John E. et. al., eds. 1999).

Numerous analytical devices known to those of skill in the art may beadapted in accordance with the present invention, to detect multipleanalytes. By way of example, dipstick, lateral flow and flow-throughdevices, particularly those that are immunoassays, may be modified inaccordance herewith in order to detect and distinguish multipleanalytes. Exemplary lateral flow devices include those described in U.S.Pat. Nos. 4,818,677, 4,943,522, 5,096,837 (RE 35,306), 5,096,837,5,118,428, 5,118,630, 5,221,616, 5,223,220, 5,225,328, 5,415,994,5,434,057, 5,521,102, 5,536,646, 5,541,069, 5,686,315, 5,763,262,5,766,961, 5,770,460, 5,773,234, 5,786,220, 5,804,452, 5,814,455,5939,331, 6,306,642. Other lateral flow devices that may be modified foruse in distinguishable detection of multiple analytes in a fluid sampleinclude U.S. Pat. Nos. 4,703,017, 6,187,598, 6,352,862, 6,485,982,6,534,320 and 6,767,714. Exemplary dipstick devices include thosedescribed in U.S. Pat. Nos. 4,235,601, 5,559,041, 5,712,172 and6,790,611. It will be appreciated by those of skill in the art that theaforementioned patents may and frequently do disclose more than oneassay configuration and are likewise referred to herein for suchadditional disclosures. Advantageously, the improvements described areapplicable to various assay, especially immunoassay, configurations.

SCDs or Test Devices of the invention can be configured to be utilizedwith existing analyte detection systems. For example, an SCD of theinvention can be configured for use with an existing test device, or anexisting test device can be configured/modified pursuant to disclosuresherein for a Test Device. Some exemplary devices that can be modified insuch a fashion include dipstick, lateral flow, cartridge, multiplexed,microtiter plate, microfluidic, plate or arrays or high throughputplatforms, such as those disclosed in U.S. Pat. Nos. 4,235,601,4,632,901, 5,559,041, 5,712,172, and 6,790,6116,448,001, 4,943,522,6,485,982, 6,656,744, 6,811,971, 5,073,484, 5,716,778, 5,798,273,6,565,808, 5,078,968, 5,415,994, 6,235,539, 6,267,722, 6,297,060,7,098,040, 6,375,896, 4,818,677, 4,943,522, 5,096,837 (RE 35,306),5,096,837, 5,118,428, 5,118,630, 5,221,616, 5,223,220, 5,225,328,5,415,994, 5,434,057, 5,521,102, 5,536,646, 5,541,069, 5,686,315,5,763,262, 5,766,961, 5,770,460, 5,773,234, 5,786,220, 5,804,452,5,814,455, 5939,331, and 6,306,642. Other lateral flow devices that maybe modified for use in distinguishable detection of multiple analytes ina fluid sample include U.S. Pat. Nos. 4,703,017, 6,187,598, 6,352,862,6,485,982, 6,534,320 and 6,767,714, 7,083,912, 5,225,322, 6,780,582,5,763,262, 6,306,642, 7,109,042, 5,952,173, and 5,914,241. Exemplarymicrofluidic devices include those disclosed in U.S. Pat. No. 5,707,799,5,837,115 and WO2004/029221. Each of the preceding patent disclosures isincorporated by reference herein in its entirety.

Reader.

The systems and methods include an immunoassay device in combinationwith a reader 400, particularly a reader 400 with a built in computer,such as a reflectance and/or fluorescent based reader, and dataprocessing software employing data reduction and curve fittingalgorithms, optionally in combination with a trained neural network foraccurately determining the presence or concentration of analyte in abiological sample. As used herein, a reader refers to an instrument fordetecting and/or quantitating data, such as on test strips comprised ina Test Device 401. The data may be visible to the naked eye, but doesnot need to be visible. The methods include the steps of performing animmunoassay on a patient sample, reading the data using a reflectanceand/or fluorescent based reader and processing the resultant data usingdata processing software employing data reduction. Preferred softwareincludes curve fitting algorithms, optionally in combination with atrained neural network, to determine the presence or amount of analytein a given sample. The data obtained from the reader then can be furtherprocessed by the medical diagnosis system to provide a risk assessmentor diagnosis of a medical condition as output. In alternativeembodiments, the output can be used as input into a subsequent decisionsupport system, such as a neural network, that is trained to evaluatesuch data.

In various embodiments, the reader can be a reflectance, transmission,fluorescence, chemo-bioluminescence, magnetic or amperometry reader (ortwo or more combinations), depending on the signal that is to bedetected from the Test Device. (e.g., LRE Medical, USA). In oneembodiment, the reader comprises a receiving port designed to receive aTest Device, but where the Test Device can only be inserted into thereceiving port if a depressible (e.g., push button) means upstream ofthe sample entry aperture has been depressed allowing the Test Device tofit into the receiving port 402. Thus, in such an embodiment, the TestDevice is placed in a reader only when the contents of the solutionreservoir (e.g., wash buffer) has been released, ensuring that thesample has been “run-through” the lateral flow membrane comprised in theTest Device.

In one embodiment, the reader is a UV LED reader which detects afluorescence signal. The fluorescence signal is excited by a lightemitting diode that emits in the UV region of the optics spectrum andwithin the absorbance peak of the fluorescence signal (e.g., lanthanidelabel). The emitted fluorescence signal is detected by a photodiode andthe wavelength of the signal detected may be limited using a long passfilter which blocks stray emitted light and accepts light withwavelengths at and around the peak emission wavelength of thefluorescence emitting label. In other embodiments, the long pass filtermay be replaced by a band pass filter. Furthermore, the excitation lightmay be limited by a band pass filter.

In another embodiment, the diode is a UV laser diode. Any conventionalUV, LED or photodiode may be utilized.

In any such embodiments, the excitation source and the detector aremounted in a single machine or molded block. For simplified reading ofthe fluorescent signals generated on the test strip. In a furtherembodiment, such a machine also comprises hard standards (e.g., FIG. 33)as described herein.

In one embodiment, the axis of the excitation light is at 90 degrees tothe Test Device or test strip comprised in a Test Device. Further, theaxis of the emitted light is at an angle other than 90 degrees to thetest strip.

In one embodiment the wavelength of the excitation light is limited by ashort pass filter. In yet another embodiment the wavelength of theexcitation light is limited by a combination of band pass filter andshort pass filter. In yet a further embodiment, the wavelength of thedetected light is limited by a combination of band pass and long passfilter. The reader can be configured to detect any of the signalemitters/labels described herein. In one embodiment, the label is any ofthe lanthanides described herein. In a further embodiment, thelanthanide used is Europium.

As indicated herein, in one embodiment, the reader is configured tocomprise one or more hard standards. Thus, the reader can be machined toprovide a implement (e.g., a jig) to hold 0.5, 0.75, 1, 1.25, 1.5, 1.75,2, 2.25, 2.5 or 3 mm standards (e.g., encased in acrylic as describedherein), which standard is disposed on about 3, 4, 5, or 6 mm centers.(e.g., See FIGS. 33A, 33B, 34).

In one embodiment, the reader is adapted with a receiving port for theTest Device FIG. 27, which itself is configured with a safeguard FIG.27C. In one embodiment the reader will accept but not process the TestDevice 401 if the push button has not been depressed, or the reader willaccept and read the Test Device, but will reject the result if the WashBuffer control does not yield a positive signal, etc. In this latterembodiment, a wash/running buffer disposed in a compartment/sac disposedupstream of the sample FIG. 27C can contain a control signal (e.g.,label emitting at a different wavelength) which the reader is programmedto detect.

The signal obtained by the reader is processed using data processingsoftware employing data reduction and curve fitting algorithms,optionally in combination with a trained neural network, to give eithera positive or negative result for each test linie, or a quantitativedetermination of the concentration of each analyte in the sample, whichis correlated with a result indicative of a risk or presence of adisease or disorder. This result can optionally be input into a decisionsupport system, and processed to provide an enhanced assessment of therisk of a medical condition as output. In one embodiment, the entireprocedure may be automated and/or computer-controlled.

Multianalyte Point of Care System.

Rapid influenza tests have been marketed for years. Most of these testsare lateral flow immunoassay tests using either gold or latex as thevisualization agent. While most of new rapid immunoassays are able todifferentiate influenza Type A from influenza Type B, only few of themhave both test lines for type A and type B on the one strip. However,none of these tests are designed to differentiate subtypes of influenzatype A. Therefore these tests may be able to detect avian influenza,none of them can tell if a patient is infected by a seasonal flu A virusor a more severe Type A subtype such as H5N1 termed avian influenza (orcurrent potential pandemic subtype of influenza A). The presentinvention is designed on concepts that when applied are to yield ahighly sensitive assay with improved reproducibility, able to detecttype A, type B and differentiate subtype H5N1 from seasonal flu(subtypes H1 and H3) and is easy to use. Efforts have been made to applymultiple new technologies with a new device design, such as pre-mixingof the sample with the conjugate, use of a chasing or wash buffer toreduce background, employ a unique generic capture reagent pRNA whichallows multiple analytes detection at high sensitivity, fluorescentlabel which is highly sensitive, etc. The combination of theseapproaches enables a novel and highly effective influenza rapid testthat is much more sensitive, provides low cost production, ease ofoperate and has the ability to differentiate seasonal flu from pandemicavian flu H5N1.

In one embodiment, the combination of features described herein areresponsible for the excellent sensitivity and reproducibility of assaysconstructed in accordance with the invention to use the novel system,which serves to concentrate ligand from a test sample at a test site thetest strip, and the use of a metal sol or other colored particle as amarker system which permits persistent visual observation of thefluorescence over a period of one to several hours beyond the minimumtime needed to complete the assay). Background noise is reduced whilemaintaining excellent sensitivity by including in the test implement acontrollably released buffer that functions to wash away excess/unboundlabel. Furthermore, one or more control sites whose color is comparedwith the test site. In some embodiments, a filtration means is comprisedin the sample implement or in the test implement, which filtration meanslimits the introduction to the test site of contaminants from the crudeor unprocessed biological sample. In yet other embodiments, thefiltration means is a membrane and includes detection and capture probesdisposed thereon 604.

Assay Methods.

In one embodiment, an assay method comprises the steps of applying thesampling implement to a subject or subject's biological sample, tocollect a sample (e.g., swabbing inside the nose, mouth, throat, ear;applying a sampling element to a biological sample obtained from asubject), inserting the collection implement into the sample collectiondevice housing chamber, squeezing the upper chamber to break open thesnap-valve and allowing a buffer to run down to the sampling implement,thus immersing the biological sample disposed thereon and running themixture of buffer and sample into a reaction chamber (e.g., lowerchamber) where a plurality of capture and detection probes bind to theirspecific target analyte. Subsequently or concurrently, the mixture isexpelled from the distal end of the SCD into a Test Device comprisingimmobilized capture moieties designed to capture a complex of analyteand detection/capture probe, via the complementary capture moiety linkedto a capture probe. Thus, a particular capture probe is designed to becomplementary to an immobilized capture moiety for one particularanalyte. Furthermore, as disclosed herein, capture moieties are disposedon a lateral flow membrane in distinct positions/patterns, where asingle line or spot(s) if detected via the signal emitting label, allowsqualitative and/or quantitative detection of a particular analyte.Therefore, by patterning particular capture probes on the lateral flowmembrane, an assay method can detect a panel of the same or relatedinfectious agent or even unrelated infectious agents, as disclosedherein.

In some embodiments a sandwich immunoassay format is utilized but anyconventional format, including a competitive assay, may be used.Typically, an indirect capture of the formed immunocomplex is utilizedin the sandwich format. One or more analytes in the sample are contactedwith one or more pairs of a detection probe and a capture probe. Eachpair contains the detection probe which is a conjugate comprising alabel and a specific binding agent (SBA) capable of specifically bindingto an analyte and a capture probe which is a conjugate comprising adetection moiety and another SBA capable of specifically binding thesame analyte. Examples of specific binding agent(s) include antibodies,aptamers, In one embodiment, each of two SBAs are specific bindingpartners in that each bind the same target antigen or analyte. Examplesof SBAs are known in the art and include but are not limited toantibodies, aptamers or oligonucleotides. In the sandwich assay, theanalyte is simultaneously bound by both the detection probe and thecapture probe. The detection moiety is part of a specific binding pairand the other partner of the pair is immobilized on the test device tocapture the immunocomplex as it flows through the test device. The useof different capture moiety pairs for each different analyte permits thedetection of multiple analyte on one test device from a single sampleand reaction sequence. In most cases, the labels for each analyte aredifferent. However, by having a specific location for each analyte as acapture zone with distinct capture moiety pairs for each analyte, it ispossible to utilize the same label for all of the analyte.

Examples of sandwich immunoassays performed on test strips are describedin U.S. Pat. Nos. 4,168,146 and 4,366,241, each of which is incorporatedherein by reference. Examples of competitive immunoassay devices arethose disclosed by U.S. Pat. Nos. 4,235,601, 4,442,204 and 5,208,535,each of which is incorporated herein by reference. Some additionalexemplary devices that can be adapted to incorporate one or morecomponents of the present invention include dipstick, lateral flow,cartridge, multiplexed, microtiter plate, microfluidic, plate or arraysor high throughput platforms, such as those disclosed in U.S. Pat. Nos.6,448,001, 4,943,522, 6,485,982, 6,656,744,6,811,971, 5,073,484,5,716,778, 5,798,273, 6,565,808, 5,078,968, 5,415,994, 6,235,539,6,267,722, 6,297,060, 7,098,040, 6,375,896, 7,083,912, 5,225,322,6,780,582, 5,763,262, 6,306,642, 7,109,042, 5,952,173, and 5,914,241.Exemplary microfluidic devices include those disclosed in U.S. Pat. No.5,707,799 and WO2004/029221.

In one such scheme, a test strip contains an enzyme labeled, mobilebinding partner for an analyte which is in a zone downstream from asample application zone. If an analyte is present in the test sample, itwill combine with its labeled binding partner to form a complex whichwill flow along the strip to a detection zone which contains a substratefor the enzyme label and capable of providing a detectable enzymaticresponse, e.g., color response in the presence of the enzyme. The stripalso contains a zone in which a predetermined amount of analyte isimmobilized, so that any labeled binding partner which does not combinewith the analyte, due to the absence of the analyte in a sample, will becaptured and thereby inhibited from reaching the detection zone.

Alternatively the strip can contain a binding partner bears a moietyhaving a detectable physical property. For example, the labeled bindingpartner can include one or more moieties, e.g., chemical groupsdetectable on the basis of certain physical properties including withoutlimitation colored species of fluorescers, phosphorescent molecules,radioisotopes, electroactive moieties, particles such as gold, etc.

In another scheme, the detection is based on the competition between aspecific analyte, e.g., ligand, antigen or antibody, the amount of whichis to be determined in a sample, and a known amount of tracer, which isgenerally the analyte or appropriate analog thereof in labeled form,with the analyte and tracer competing for a limited number of availablebinding sites on a binder which is specific towards the analyte andtracer. For example, if the concentration of tracer and binder is fixedand the only variable is the level of analyte, then the unknown level ofanalyte can be measured by determining the amount of bound and/or freetracer in the system. The values determined in the assay are comparedwith the values given by a range of known amounts of the analyte treatedin the same manner, and by such comparison, one can determine the amountof analyte in the sample.

In general, the tracers used in such assays require eitherinstrumentation and/or treatment of the tracer in order to determine thetracer in the bound and/or free portion of the assay as a measure ofanalyte. For example, in an assay in which an enzyme is used as thelabel or marker for the tracer, the enzyme must be developed with asuitable developer. When the label or marker is a fluorescent material,the tracer in the bound and/or free portion is determined by the use ofappropriate instrumentation for determining fluorescence.

Alternatively the tracer used in the assay is a ligand labeled with aparticulate label which is visible when bound to the binder on thesupport or when bound to the analyte bound to the binder on the support,without further treatment, and wherein the ligand is bound by either thebinder or analyte. See also U.S. Pat. No. 4,703,017, which isincorporated herein by reference.

In another particular aspect, the non-nucleic acid based screening testof the present invention includes any solid phase, lateral flow, orflow-through tests. In general, solid phase immunoassay devicesincorporate a solid support to which one member of a ligand-receptorpair, usually an antibody, antigen, or hapten, is bound. Common earlyforms of solid supports were plates, tubes, or beads of polystyrene,which were known from the fields of radioimmunoassay and enzymeimmunoassay. More recently, a number of porous materials such as nylon,nitrocellulose, cellulose acetate, glass fibers, and other porouspolymers have been employed as solid supports

In one embodiment, a sample is collected from a subject via a samplingimplement 102 and placed back into the cylinder housing of the SCDdevice 200. The SCD can first be inserted into a Test Device, or priorto insertion into a Test Device, a solution contained in the uppersealed chamber of the SCD is released to effect washing the sample andsolution into a mixture downwards into a reaction chamber. In thereaction chamber is disposed either liquid or solid reagents comprisingdetection and capture probes that target one or more different analytesas disclosed herein, thereby forming a complex of analyte bound todetection and capture probe. The sample is then expelled from the SCDinto a Test Device through an aperture that seals the contact betweenthe SCD and the Test Device from the outside environment (e.g.,preventing any spillage, aerosol or contamination). The sample mixturecan flow as a result of gravity or the force of air pressure produced bysqueezing the SCD (e.g., upper sealed chamber), into a Test Device. Thesample is driven by capillary force and/or by wash buffer comprised inthe Test Device 307 so as to allow any analyte-probe complex to passthrough the lateral flow membrane contained in the Test Device. Captureprobes and complementary immobilized capture moieties bind or hybridizeto each other in predetermined lines or spots on the lateral flowmembrane 803 or 804, whereby detection probes (via conjugate labelscontained thereon) will provide a detectable signal which cansubsequently be read to determine which analytes were present in thesample processed.

In one embodiment, Test Devices with samples processed thereon, can beset aside for time periods of about 1, 2, 3, 4, 5, 6 or 8 hours beforereading the results, and yet provide results as accurately as if read in15 or 20 minutes after processing. Thus, the signals produced are stablefor long periods of time so that reading the results may occur at asignificantly later time after the tests are actually performed. This isa great improvement for point-of-care diagnostics, where in the fieldconditions often present limited resources in manpower and time, andwhere the test setting can be in remote regions that are not easily orquickly accessed.

Binding Reagents.

One aspect of the invention is directed to binding reagents disposed inthe SCD. For example, in some immunoassays, an antibody pair isutilized, where each member of the pair can specifically bind the sametarget analyte, wherein one antibody is a capture antibody and the otheris a detection antibody. A capture antibody is linked, directly orindirectly, to a capture moiety which is “captured” by a cognateimmobilized capture moiety disposed on a solid support (e.g.,nitrocellulose membrane). Furthermore, the detection antibody (i.e.,detection probe) is linked to a detectable label. The detection antibodyis preferably labeled by conjugation to a physically detectable label,and upon contacting with the sample containing the target analyte formsa complex. The antibody-analyte complex can then be immobilized on asolid support via the capture moiety. The resulting complex immobilizedon the solid support, is detectable by virtue of the label.

In one embodiment, the SCD reagent solution or solid substrate comprisesa plurality of different detection probes, each detection probe capableof binding to a different target and each detection probe being labeledwith or enabling the formation of a detection signal so that thepresence of each target is indicated by the formation of a signal at thetest zone for that target (i.e., in the Test Device); wherein the targetfor at least two of the capture moieties is an infectious agent or adisease causing micro-organism or a marker indicating the existence of adisease, disorder, or condition of the host from which the samplesolution was derived, and wherein at least two of the capture moietiesare capable of binding to different components or markers of the sameinfectious agent or disease causing microorganism, or to differentmarkers for the same disease, disorder, or condition not caused by aninfectious agent or disease causing microorganism, as targets for thosecapture moieties. Furthermore, the SCD will also comprise a plurality ofdifferent capture probes, each of which is paired up with a detectionprobe, where the pairing is defined by the capability to bind aparticular target analyte.

As used herein, the term “specifically binds” refers to the bindingspecificity of a specific binding pair. “Specific binding pair member”refers to a member of a specific binding pair (“sbp”), which means twodifferent molecules wherein one of the molecules specifically binds withthe second molecule through chemical or physical means. For example, apair of pRNAs or an aptamer/target antigen pair, or streptavidin-biotinprovide exemplary specific binding pair members or sbp. The twomolecules are related in the sense that their binding with each other issuch that they are capable of distinguishing their binding partner fromother assay constituents having similar characteristics. The members ofthe specific binding pair are referred to as ligand and receptor(antiligand), sbp member and sbp partner, and the like. A molecule mayalso be a sbp member for an aggregation of molecules; for example anantibody raised against an immune complex of a second antibody and itscorresponding antigen may be considered to be an sbp member for theimmune complex.

In addition to antigen and antibody specific binding pair members, otherspecific binding pairs include, as examples without limitation, biotinand avidin, carbohydrates and lectins, complementary nucleotidesequences, complementary peptide sequences, effector and receptormolecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes,a peptide sequence or chemical moiety (such as digoxin/anti-digoxin) andan antibody specific for the sequence, chemical moiety or the entireprotein, polymeric acids and bases, dyes and protein binders, peptidesand specific protein binders (e.g., ribonuclease, S-peptide andribonuclease S-protein), metals and their chelators, and the like.Furthermore, specific binding pairs can include members that are analogsof the original specific binding member, for example an analyte-analogor a specific binding member made by recombinant techniques or molecularengineering.

An sbp member is analogous to another sbp member if they are bothcapable of binding to another identical complementary sbp member. Suchan sbp member may, for example, be either a ligand or a receptor thathas been modified by the replacement of at least one hydrogen atom by agroup to provide, for example, a labeled ligand or labeled receptor. Thesbp members can be analogous to or complementary to the analyte or to ansbp member that is complementary to the analyte. If the specific bindingmember is an immunoreactant it can be, for example, an antibody,antigen, hapten, or complex thereof. If an antibody is used, it can be amonoclonal or polyclonal antibody, a recombinant protein or antibody, achimeric antibody, a mixture(s) or fragment(s) thereof, as well as amixture of an antibody and other specific binding members. Otherexamples of binding pairs that can be incorporated into the detectionmolecules are disclosed in U.S. Pat. Nos. 6,946,546, 6,967,250,6,984,491, 7,022,492, 7,026,120, 7,022,529, 7,026,135, 7,033,781,7,052,854, 7,052,916 and 7,056,679.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, andincludes any immunoglobulin, including monoclonal antibodies, polyclonalantibodies, multispecific or bispecific antibodies, that bind to aspecific antigen. A complete antibody comprises two heavy chains and twolight chains. Each heavy chain consists of a variable region and afirst, second, and third constant region, while each light chainconsists of a variable region and a constant region. The antibody has a“Y” shape, with the stem of the Y consisting of the second and thirdconstant regions of two heavy chains bound together via disulfidebonding. Each arm of the Y consists of the variable region and firstconstant region of a single heavy chain bound to the variable andconstant regions of a single light chain. The variable regions of thelight and heavy chains are responsible for antigen binding. The variableregion in both chains generally contains three highly variable loopscalled the complementarity determining regions (CDRs) (light (L) chainCDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs includingHCDR1 , HCDR2, HCDR3) (as defined by Kabat, et al., Sequences ofProteins of Immunological Interest, Fifth Edition (1991), vols. 1-3, NIHPublication 91-3242, Bethesda Md.). The three CDRs are interposedbetween flanking stretches known as framework regions (FRs), which aremore highly conserved than the CDRs and form a scaffold to support thehypervariable loops. The constant regions of the heavy and light chainsare not involved in antigen binding, but exhibit various effectorfunctions. The recognized immunoglobulin genes include the kappa,lambda, alpha, gamma, delta, epsilon, and mu constant regions, as wellas myriad immunoglobulin variable region genes. Light chains areclassified as either kappa or lambda. Heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes and subclasses include IgG, IgG1, IgG2, IgG3,IgG4, IgM, IgA, IgA1, or IgA2, IgD, and IgE, respectively. Typically, anantibody is an immunoglobulin having an area on its surface or in acavity that specifically binds to and is thereby defied as complementarywith a particular spatial and polar organization of another molecule.The antibody can be polyclonal or monoclonal. Antibodies may include acomplete immunoglobulin or fragments thereof. Fragments thereof mayinclude Fab, Fv and F(ab′)2, Fab′, and the like. Antibodies may alsoinclude chimeric antibodies or fragment thereof made by recombinantmethods. The term “antibody” as used herein Antibodies are assigned toclasses based on the amino acid sequence of the constant region of theirheavy chain. The major classes of antibodies are IgA, IgD, IgE, IgG, andIgM, with several of these classes divided into subclasses such as.

In addition to an intact immunoglobulin, the term “antibody” as usedherein further refers to an immunoglobulin fragment thereof (i.e., atleast one immunologically active portion of an immunoglobulin molecule),such as a Fab, Fab′, F(ab′)₂, Fv fragment, a single-chain antibodymolecule, a multispecific antibody formed from any fragment of animmunoglobulin molecule comprising one or more CDRs. In addition, anantibody as used herein may comprise one or more CDRs from a particularhuman immunoglobulin grafted to a framework region from one or moredifferent human immunoglobulins.

“Fab” with regards to an antibody refers to that portion of the antibodyconsisting of a single light chain (both variable and constant regions)bound to the variable region and first constant region of a single heavychain by a disulfide bond.

“Fab′” refers to a Fab fragment that includes a portion of the hingeregion.

“F(ab′)₂ refers to a dimer of Fab′.

“Fc′” with regards to an antibody refers to that portion of the antibodyconsisting of the second and third constant regions of a first heavychain bound to the second and third constant regions of a second heavychain via disulfide bonding. The Fc portion of the antibody isresponsible for various effector functions but does not function inantigen binding.

“Fv” with regards to an antibody refers to the smallest fragment of theantibody to bear the complete antigen binding site. An Fv fragmentconsists of the variable region of a single light chain bound to thevariable region of a single heavy chain.

“Single-chain Fv antibody” or “scFv” refers to an engineered antibodyconsisting of a light chain variable region and a heavy chain variableregion connected to one another directly or via a peptide linkersequence (Houston 1988).

“Single-chain Fv-Fc antibody” or “scFv-Fc” refers to an engineeredantibody consisting of a scFv connected to the Fc region of an antibody.

The term “epitope” as used herein refers to the group of atoms and/oramino acids on an antigen molecule to which an antibody binds.

The term “monoclonal antibody” as used herein refers to an antibody or afragment thereof obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single epitope on the antigen. Monoclonalantibodies are in contrast to polyclonal antibodies which typicallyinclude different antibodies directed against different epitopes on theantigens. Although monoclonal antibodies are traditionally derived fromhybridomas, the monoclonal antibodies of the present invention are notlimited by their production method. For example, the monoclonalantibodies of the present invention may be made by the hybridoma methodfirst described by Kohler at al., Nature, 256:495 (1975), or may be madeby recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

The term “chimeric antibody” as used herein refers to an antibody inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from another species or belonging to another antibody class orsubclass, as well as fragments of such an antibody, so long as suchfragments exhibit the desired antigen-binding activity (U.S. Pat. No.4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci.USA, 81:6851 6855 (1984)).

The term “humanized antibody” used herein refers to an antibody orfragments thereof which are human immunoglobulins (recipient antibody)in which residues from part or all of a CDR of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinity,and capacity. In some instances, FR residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues which are found neither in therecipient antibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin Fc region, typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature, 321:522 525 (1986); Reichmannet al., Nature, 332:323 329 (1988); Presta, Curr. Op. Struct. Biol.,2:593 596 (1992); and Clark, Immunol. Today 21: 397 402 (2000).

The present invention provides anti-H5 monoclonal antibodies that areproduced by mice hybridoma cell strains 8H5, 3C8, 10F7, 4D1, 3G4 and2F2, These monoclonal antibodies are named after the hybridoma cellstrains that produce them. Thus the anti-H5 monoclonal antibodies thatare produced by mice hybridoma cell strains 8H5, 3C8, 10F7, 4D1, 3G4,and 2F2, respectively, are named monoclonal antibodies 8H5, 3C8, 10F7,4D1, 3G4, and 2F2, respectively. Monoclonal antibodies 8H5, 3C8, 10F7,4D1, 3G4, and 2F2 specifically bind to the hemagglutinin of subtype H5avian influenza virus. The mice hybridoma cell strains 8H5, 3C8, 10F7,4D1, 3G4, and 2F2 were deposited in China Center for Typical CultureCollection (CCTCC, Wuhan University, Wuhan, China) on Jan. 17, 2006 withdeposit numbers of CCTCC-C200607 (hybridoma cell strain 8H5),CCTCC-C200605 (hybridoma cell strain 3C8), CCTCC-C200608 (hybridoma cellstrain 10F7), CCTCC-C200606 (hybridoma cell strain 4D1), CCTCC-C200604(hybridoma cell strain 3G4) and CCTCC-C200424 (hybridoma cell strain2F2).

In various embodiment, monoclonal antibodies are provided that block thebinding of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 304, or 2F2 to thehemagglutinin of subtype H5 avian influenza virus. Such blockingmonoclonal antibodies may bind to the same epitopes on the hemagglutininthat are recognized by monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4,or 2F2. Alternatively, those blocking monoclonal antibodies may bind toepitopes that overlap sterically with the epitopes recognized bymonoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4, or 2F2. These blockingmonoclonal antibodies can reduce the binding of monoclonal antibodies8H5, 3C8, 10F7, 4D1, 3G4, or 2F2 to the hemagglutinin of subtype H5avian influenza virus by at least about 50%. Alternatively, they mayreduce binding by at least about 60%, preferably at least about 70%,more preferably at least about 75%, more preferably at least about 80%,more preferably at least about 85%, even more preferably at least about90%, even more preferably at least about 95%, most preferably at leastabout 99%.

The ability of a test monoclonal antibody to reduce the binding of aknown monoclonal antibody to the H5 hemagglutinin may be measured by aroutine competition assay such as that described in Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988). For example, such an assay could be performed bypre-coating a microtiter plate with antigens, incubating the pre-coatedplates with serial dilutions of the unlabeled test antibodies admixedwith a selected concentration of the labeled known antibodies, washingthe incubation mixture, and detecting and measuring the amount of theknown antibodies bound to the plates at the various dilutions of thetest antibodies. The stronger the test antibodies compete with the knownantibodies for binding to the antigens, the more the binding of theknown antibodies to the antigens would be reduced. Usually, the antigensare pre-coated on a 96-well plate, and the ability of unlabeledantibodies to block the binding of labeled antibodies is measured usingradioactive or enzyme labels.

Monoclonal antibodies may be generated by the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975). In the hybridomamethod, a mouse or other appropriate host animal is immunized by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in thehost animal by multiple subcutaneous or intraperitoneal injections. Itmay be useful to conjugate the immunizing agent to a protein known to beimmunogenic in the host animal being immunized, such as serum albumin,or soybean trypsin inhibitor. Examples of adjuvants which may beemployed include Freund's complete adjuvant and MPL-TDM. Afterimmunization, the host animal makes lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theantigen used for immunization. Alternatively, lymphocytes may beimmunized in vitro. Desired lymphocytes are collected and fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59 103, Academic Press, 1996).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOP-21 and MC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63,Marcel Dekker, Inc., New York, 1987).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107: 220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the cells may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103,Academic Press, 1996). Suitable culture media for this purpose include,for example, DMEM or RPMI-1640 medium. In addition, the hybridoma cellsmay be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies of the invention may also be made by conventionalgenetic engineering methods. DNA molecules encoding the heavy and lightchains of the monoclonal antibodies may be isolated from the hybridomacells, for example through PCR using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies. Then the DNA molecules are insertedinto expression vectors. The expression vectors are transfected intohost cells such as E. coil cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein. The host cells are cultured under conditionssuitable for the expression of the antibodies.

The antibodies of the invention can bind to the H5 hemagglutinin withhigh specificity and affinity. The antibodies shall have lowcross-reactivity with other subtypes of hemagglutinin, preferably nocross-reactivity with other subtypes of hemagglutinins. In one aspect,the invention provides antibodies that bind to H5 hemagglutinin with aK_(D) value of less than 1×10⁻⁵M. Preferably, the K_(D) value is lessthan 1×10⁻⁶M. More preferably, the K_(D) value is less than 1×10⁻⁷M.Most preferably, the K_(D) value is less than 1×10⁻⁸M.

The antibodies of the invention may contain the conventional “Y” shapestructure comprised of two heavy chains and two fight chains. Inaddition, the antibodies may also be the Fab fragment, the Fab′fragment, the F(ab)₂ fragment or the Fv fragment, or another partialpiece of the conventional “Y” shaped structure that maintains bindingaffinity to the hemagglutinin. The binding affinity of the fragments tohemagglutinin may be higher or lower than that of the conventional “Y”shaped antibodies.

The antibody fragments may be generated via proteolytic digestion ofintact antibodies (see, e.g., Morimoto et al., J. Biochem. Biophys.Methods, 24:107-117, (1992) and Brennan et al., Science, 229:81 (1985)).Additionally, these fragments can also be produced directly byrecombinant host cells (reviewed in Hudson, Curr. Opin. Immunol., 11:548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). Forexample, Fab′ fragments can be directly recovered from E. coil andchemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology, 10:163 167 (1992)). In another embodiment, the F(ab′),is formed using the leucine zipper GCN4 to promote assembly of theF(ab′)₂ molecule. According to another approach, Fv, Fab or F(ab′)₂fragments can be isolated directly from recombinant host cell culture.Other techniques for the production of antibody fragments will beapparent to a person with ordinary skill in the art.

The present invention provides isolated nucleic acid molecules encodingantibodies or fragments thereof that specifically bind to H5hemagglutinin. Nucleic acid molecules encoding the antibodies can beisolated from hybridoma cells. The nucleic acid sequences of themolecules can be determined using routine techniques known to a personwith ordinary skill in the art. Nucleic acid molecules of the inventioncan also be prepared using conventional genetic engineering techniquesas well as chemical synthesis. In one aspect, the present inventionprovides an isolated nucleic acid molecule encoding the variable regionof the heavy chain of an anti-H5 (HA) antibody or a portion of thenucleic acid molecule. In another aspect, the present invention providesan isolated nucleic acid molecule encoding the variable region of thelight chain of an anti-H5 (HA) antibody or a portion of the nucleic acidmolecule. In another aspect, the present invention provides an isolatednucleic acid molecule encoding the CDRs of the antibody heavy chain orlight chain variable regions.

In one aspect, the present invention provides isolated nucleic acidmolecules encoding the variable regions of the heavy chain and lightchain of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4, and 2F2. Thenucleic acid sequences encoding the heavy chain variable regions ofmonoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4, and 2F2 are set forth inSEQ ID N0: 1, SEQ ID NO: 5, SEQ ID N0: 9, SEQ ID NO:16, SEQ ID NO:20 andSEQ ID NO: 24, respectively. The nucleic acid sequences encoding thelight chain variable regions of monoclonal antibodies 8H5, 3C8, 10F7,4D1, and 2F2 are set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID N0: 11,SEQ ID NO:18, SEQ ID NO: 26, respectively. The present invention alsoincludes degenerative analogs of the nucleic acid molecules encoding thevariable regions of the heavy chain and light chain of monoclonalantibodies 8H5, 3C8, 10F7, 4D1, 3G4 and 2F2.

In another aspect, the present invention provides isolated nucleic acidvariants that share sequence identity with the nucleic acid sequences ofSEQ ID N0: 1, SEQ ID N0: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID N0: 9,SEQ ID N0: 11, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24or SEQ ID NO:26. In one embodiment, the nucleic acid variants share atleast 70% sequence identity, preferably at least 75% sequence identity,more preferably at least 80% sequence identity, more preferably at least85% sequence identity, more preferably at least 90% sequence identity,most preferably at least 95% sequence identity, to the sequences of SEQID N0: 1, SEQ ID N0: 3, SEQ ID NO: 5, SEQ II) NO: 7, SEQ ID N0: 9, SEQID N0: 11, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:24 or SEQID NO:26.

The present invention also provides isolated nucleic acid moleculesencoding antibody fragments that are still capable of specificallybinding to subtype H5 of avian influenza virus.

The present invention further provides isolated nucleic acid moleculesencoding an antibody heavy chain variable region comprising the aminoacid sequence set forth in SEQ ID NOs: 28-30, SEQ ID NOs: 34-36, SEQ IDNOs: 40-42, SEQ ID NOs: 46-48; SEQ ID NOs: 52-54, and SEQ ID NOs: 58-60.The present invention provides isolated nucleic acid molecules encodingan antibody light chain variable region comprising the amino acidsequence set forth in SEQ ID NOs: 31-33, SEQ ID NOs: 37-39, SEQ ID NOs:43-45, SEQ ID NOs: 49-51, SEQ ID NOs: 55-57, and SEQ ID NOs: 61-63.

The present invention provides recombinant expressing vectors comprisingthe isolated nucleic acid molecules of the invention. It also provideshost cells transformed with the nucleic acid molecules. Furthermore, thepresent invention provides a method of producing antibodies of theinvention comprising culturing the host cells under conditions whereinthe nucleic acid molecules are expressed to produce the antibodies andisolating the antibodies from the host cells.

Antibody Polypeptide Sequences

The amino acid sequences of the variable regions of the heavy chain andlight chain of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4 and 2F2have been deduced from their respective nucleic acid sequences. Theamino acid sequences of the heavy chain variable regions of monoclonalantibodies 8H5, 3C8, 10F7, 4D1, 3G4 and 2F2 are set forth in SEQ IDN0:2, SEQ ID N0: 6, SEQ ID N0: 10, SEQ ID NO:17, SEQ ID NO:21, and SEQID NO:25, respectively. The amino acid sequences of the light chainvariable regions of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, and 2F2are set forth in SEQ ID N0:4, SEQ ID N0:8, SEQ ID NO:12, SEQ ID NO:19,and SEQ ID NO:27. In one aspect, the present invention provides anti-H5antibodies comprising a heavy chain variable region comprising the aminoacid sequences as set forth in SEQ ID N0:2, SEQ ID N0:6, SEQ ID NO: 10,SEQ ID NO:17, SEQ ID NO:21, and SEQ ID NO:25. In another aspect, thepresent invention provides anti-H5 antibodies comprising a light chainvariable region comprising the amino acid sequences as set forth in SEQID N0:4, SEQ ID N0:8, SEQ ID NO:12, SEQ ID NO:19, and SEQ ID NO:27.

In another aspect, the present invention provides an antibody heavychain comprising a variable region having at least 70% sequenceidentity, preferably at least 75% sequence identity, more preferably atleast 80% sequence identity, more preferably at least 85% sequenceidentity, more preferably at least 90% sequence identity, mostpreferably at least 95% sequence identity to the amino acid sequencesset forth in SEQ ID N0:2, SEQ ID N0:6, SEQ ID N0: 10, SEQ ID NO:17, SEQID NO:21, and SEQ ID NO:25.

In another aspect, the present invention provides an antibody lightchain comprising a variable region having at least 70% sequenceidentity, preferably at least 75% sequence identity, more preferably atleast 80% sequence identity, more preferably at least 85% sequenceidentity, more preferably at least 90% sequence identity, mostpreferably at least 95% sequence identity to the amino acid sequencesset forth in SEQ ID N0:4, SEQ ID N0:8, SEQ ID NO:12, SEQ ID NO:19, andSEQ ID NO:27.

The amino acid sequences of the CDRs of the variable regions of theheavy chain and light chain of monoclonal antibodies 8H5, 3C8, 10F7,4D1, 3G4, and 2F2 have also been determined as follows:

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain ofmonoclonal antibody 8H5 are set forth in SEQ ID Nos:28-30, respectively.The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain ofmonoclonal antibody 8H5 are set forth in SEQ ID Nos:31-33, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain ofmonoclonal antibody 3C8 are set forth in SEQ ID Nos:34-36, respectively.The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain ofmonoclonal antibody 3C8 are set forth in SEQ ID Nos:37-39, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain ofmonoclonal antibody 10F7 are set forth in SEQ ID Nos:40-42,respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of thelight chain of monoclonal antibody 10F7 are set forth in SEQ IDNos:43-45, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain ofmonoclonal antibody 4D1 are set forth in SEQ ID Nos:46-48, respectively.The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain ofmonoclonal antibody 4D1 are set forth in SEQ ID Nos:49-51, respectively,

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain ofmonoclonal antibody 3G4 are set forth in SEQ ID Nos:52-54, respectively.The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain ofmonoclonal antibody 3G4 are set forth in SEQ ID Nos:55-57, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain ofmonoclonal antibody 2F2 are set forth in SEQ ID Nos:58-60, respectively.The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain ofmonoclonal antibody 2F2 are set forth in SEQ ID Nos:61-63, respectively.

TABLE 1 Six strains of monoclonal antibody CDRs amino acid sequence.Monoclonal Antibody heavy chain Antibody light chain antibody CDRs aminoacid sequence CDRs amino acid sequence strains CDR1 CDR2 CDR3 CDR1 CDR2CDR3 8H5 GYTFSNYW ILPGSDRT ANRYDGYYFGLDY SSVNF YSS QHFTSSPYT (SEQ ID NO:28) (SEQ ID NO: 29) (SEQ ID NO: 30) (SEQ ID (SEQ ID (SEQ ID NO: NO: 31)NO: 32) 33) 3C8 GYSFTNYG INTHTGEP ARWNRDAMDY ESVDSSDNSL RAS QQSIGDPPYT(SEQ ID NO: 34) (SEQ ID NO: 35) (SEQ ID NO: 36) (SEQ ID (SEQ ID (SEQ IDNO: NO: 37) NO: 38) 39) 10F7 GYTFTSYW IDPSDSYT ARGGTGDFHYAMDY QGISSN HGTQYVQFPYT (SEQ ID NO: 40) (SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID (SEQ ID(SEQ ID NO: NO: 43) NO: 44) 45) 4D1 GYTFTSYW IDPSDSFT ARGGPGDFRYAMDYQGISSN HGT VQYVQFPYT (SEQ ID NO: 46) (SEQ ID NO: 47) (SEQ ID NO: 48)(SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 50) 51) 3G4 GYTFTDYA INTDYGDTARSDYDYYFCGMDY (SEQ ID (SEQ ID (SEQ ID NO: (SEQ ID NO: 52) (SEQ ID NO:53) (SEQ ID NO: 54) NO: 55) NO: 56) 57) 2F2 GFSLTGYG IWAEGRTAREVITTEAWYFDV QSISDY YAS QNGHTFPLT (SEQ ID NO: 58) (SEQ ID NO: 59) (SEQID NO: 60) (SEQ ID (SEQ ID (SEQ ID NO: NO: 61) NO: 62) 63)

In another aspect, the present invention provides an anti-H5 monoclonalantibody heavy chain or a fragment thereof, comprising the followingCDRs: (i) one or more CDRs selected from SEQ ID NOs: 28-30; (ii) one ormore CDRs selected from SEQ ID NOs: 34-36; (iii) one or more CDRsselected from SEQ ID NOs: 40-42; (iv) one or more CDRs selected from SEQID NOs: 46-48; (v) one or more CDRs selected from SEQ ID NOs: 52-54; or(vi) one or more CDRs selected from SEQ ID NOs: 58-60. In oneembodiment, the anti-H5 monoclonal antibody heavy chain or a fragmentthereof comprises three CDRs having the amino acid sequences set forthin SEQ ID NOs: 28-30, respectively. In another embodiment, the anti-H5monoclonal antibody heavy chain or a fragment thereof comprises threeCDRs having the amino acid sequences set forth in SEQ ID NOs: 34-36,respectively. In another embodiment, the anti-H5 monoclonal antibodyheavy chain or a fragment thereof comprises three CDRs having the aminoacid sequences set forth in SEQ ID NOs: 40-42. in another embodiment,the anti-H5 monoclonal antibody heavy chain or a fragment thereofcomprises three CDRs having the amino acid sequences set forth in SEQ IDNOs: 46-48. In another embodiment, the anti-H5 monoclonal antibody heavychain or a fragment thereof comprises three CDRs having the amino acidsequences set forth in SEQ ID NOs: 52-54. in another embodiment, theanti-H5 monoclonal antibody heavy chain or a fragment thereof comprisesthree CDRs having the amino acid sequences set forth in SEQ ID NOs:58-60.

In another aspect, the CDRs contained in the anti-H5 monoclonal antibodyheavy chains or fragments thereof of the present invention may includeone or more amino acid substitution, addition and/or deletion from theamino acid sequences set forth in SEQ ID NOs: 28-30, 34-36, 40-42,46-48, 52-54, and 58-60. Preferably, the amino acid substitution,addition and/or deletion occur at no more than three amino acidpositions. More preferably, the amino acid substitution, addition and/ordeletion occur at no more than two amino acid positions. Mostpreferably, the amino acid substitution, addition and/or deletion occurat no more than one amino acid position.

In another aspect, the present invention provides an anti-H5 monoclonalantibody light chain or a fragment thereof, comprising the followingCDRs: (i) one or more CDRs selected from SEQ ID NOs: 31-33; (ii) one ormore CDRs selected from SEQ ID NOs: 37-39; (iii) one or more CDRsselected from SEQ ID NOs: 43-45; (iv) one or more CDRs selected from SEQID NOs: 49-51; (v) one or more CDRs selected from SEQ ID NOs: 55-57; or(vi) one or more CDRs selected from SEQ ID NOs: 61-63. In oneembodiment, the anti-H5 monoclonal antibody light chain or a fragmentthereof comprises three CDRs having the amino acid sequences set forthin SEQ ID NOs: 31-33, respectively. In another embodiment, the anti-H5monoclonal antibody light chain or a fragment thereof comprises threeCDRs having the amino acid sequences set forth in SEQ ID NOs: 37-39,respectively. In another embodiment, the anti-H5 monoclonal antibodylight chain or a fragment thereof comprises three CDRs having the aminoacid sequences set forth in SEQ ID NOs: 43-45. In another embodiment,the anti-H5 monoclonal antibody light chain or a fragment thereofcomprises three CDRs having the amino acid sequences set forth in SEQ IDNOs: 49-51. In another embodiment, the anti-H5 monoclonal antibody lightchain or a fragment thereof comprises three CDRs having the amino acidsequences set forth in SEQ ID NOs: 55-57. In another embodiment, theanti-H5 monoclonal antibody light chain or a fragment thereof comprisesthree CDRs having the amino acid sequences set forth in SEQ ID NOs:61-63.

In another aspect, the CDRs contained in the anti-H5 monoclonal antibodylight chains or fragments thereof of the present invention may includeone or more amino acid substitution, addition and/or deletion from theamino acid sequences set forth in SEQ ID NOs: 31-33, 37-39, 43-45,49-51, 55-57, and 61-63. Preferably, the amino acid substitution,addition and/or deletion occur at no more than three amino acidpositions. More preferably, the amino acid substitution, addition and/ordeletion occur at no more than two amino acid positions. Mostpreferably, the amino acid substitution, addition and/or deletion occurat no more than one amino acid position.

TABLE 2 The Amino Acid Sequences of the 7aa peptides that bind to 8H5mAb or 3C8 mAb. Monoclonal Antibody 7peptide sequences Sequence No. 8H5H G M L P V Y SEQ ID No: 64 P P S N Y G R SEQ ID No: 65 P P S N F G KSEQ ID No: 66 G D P W F T S SEQ ID No: 67 N S G P W L T SEQ ID No: 683C8 W P P L S K K SEQ ID No: 70 N T F R T P I SEQ ID No: 71 N T F R D PN SEQ ID No: 72 N P I W T K L SEQ ID No: 73

The variants generated by amino acid substitution, addition and/ordeletion in the variable regions of the above described antibodies orthe above described CDRs maintain the ability of specifically binding tosubtype H5 of avian influenza virus. The present inventions also includeantigen-binding fragments of such variants.

Monoclonal antibody variants of the invention may be made byconventional genetic engineering methods. Nucleic acid mutations may beintroduced into the DNA molecules using methods known to a person withordinary skill in the art. Alternately, the nucleic acid moleculesencoding the heavy and light chain variants may be made by chemicalsynthesis.

In another aspect, the screening method of the invention comprises thesteps of (i) culturing a peptide display library under conditionssuitable for peptide expression; (ii) contacting the culture solutionwith monoclonal antibodies of the invention; (iii) selecting the phageclones that specifically bind to said monoclonal antibodies. Themonoclonal antibodies used for the screening may include withoutlimitation the monoclonal antibodies 8H5, 3C8, 10F7, 4D1 and 3G4.Example 12 included herein describes in detail an assay thatsuccessfully screened short peptides that bind to the monoclonalantibodies of the invention using a peptide phage display libraries.

TABLE 3 The sequences of the 12aa peptides that bind to 8H5 mAb. Peptidesection No. Amino Acid Sequence Base Sequence 121 MEPVKKYPTRSPATGGAGCCGGTGAAGAAGTATCCGACGCGTTCTCCT (SEQ ID NO: 74) (SEQ ID NO: 75) 122ETQLTTAGLRLL GAGACTCAGCTGACTACGGCGGGTCTTCGGCTGCTT (SEQ ID NO: 76) (SEQID NO: 77) 123 ETPLTETALKWH GAGACGCCTCTTACGGAGACGGCTTTGAAGTGGCAT (SEQ IDNO: 78) (SEQ ID NO: 79) 124 QTPLTMAALELFCAGACGCCGCTGACTATGGCTGCTCTTGAGCTTTTT (SEQ ID NO: 80) (SEQ ID NO: 81) 125DTPLTTAALRLV GATACTCCGCTGACGACGGCGGCTCTTCGGCTGGTT (SEQ ID NO: 82) (SEQID NO: 83) 126 TPLTLWALSGLR ACGCCGCTTACGCTTTGGGCTCTTTCTGGGCTGAGG (SEQ IDNO: 84) (SEQ ID NO: 85) 128 QTPLTETALKWHCAGACGCCTCTTACGGAGACGGCTTTGAAGTGGCAT (SEQ ID NO: 86) (SEQ ID NO: 87) 129QTPLTMAALELL CAGACGCCTCTGACTATGGCGGCTCTTGAGCTTCTT (SEQ ID NO: 88) (SEQID NO: 89) 130 HLQDGSPPSSPH CAGACGCCTCTGACTATGGCGGCTCTTGAGCTTCTT (SEQ IDNO: 90) (SEQ ID NO: 91) 131 GHVTTLSLLSLRGGGCATGTGACGACTCTTTCTCTTCTGTCGCTGCGG (SEQ ID NO: 92) (SEQ ID NO: 93) 132FPNFDWPLSPWT TTTCCGAATTTTGATTGGCCTCTGTCTCCGTGGACG (SEQ ID NO: 94) (SEQID NO: 95) 133 ETPLTEPAFKRH GAGACGCCTCTTACGGAGCCGGCTTTTAAGCGGCAT (SEQ IDNO: 96) (SEQ ID NO: 97)

As used herein the term “Analyte” refers to the compound or compositionto be detected or measured and which has at least one epitope or bindingsite. The analyte can be any substance for which there exists anaturally occurring analyte specific binding member or for which ananalyte-specific binding member can be prepared, e.g., carbohydrate andlectin, hormone and receptor, complementary nucleic acids, and the like.Further, possible analytes include virtually any compound, composition,aggregation, or other substance which may be immunologically detected.That is, the analyte, or portion thereof, will be antigenic or haptenichaving at least one determinant site, or will be a member of a naturallyoccurring binding pair.

Analytes include, but are not limited to, toxins, organic compounds,proteins, peptides, microorganisms, bacteria, viruses, amino acids,nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs(including those administered for therapeutic purposes as well as thoseadministered for illicit purposes), pollutants, pesticides, andmetabolites of or antibodies to any of the above substances. The termanalyte also includes any antigenic substances, haptens, antibodies,macromolecules, and combinations thereof. A non-exhaustive list ofexemplary analytes is set forth in U.S. Pat. No. 4,366,241, at column19, line 7 through column 26, line 42, the disclosure of which isincorporated herein by reference. Further descriptions and listings ofrepresentative analytes are found in U.S. Pat. Nos. 4,299,916;4,275,149; and 4,806,311, all incorporated herein by reference. In someembodiments, the SCD or Test Device are configured to detect a pluralityof different analytes (e.g., FIG. 26).

Labeled Reagents

“Labeled reagent” refers to a substance comprising a detectable labelattached with a specific binding member (e.g., detection probe). Theattachment may be covalent or non-covalent binding, but the method ofattachment is not critical to the present invention. The label allowsthe label reagent to produce a detectable signal that is related to thepresence of analyte in the fluid sample. The specific binding membercomponent of the label reagent is selected to directly bind to theanalyte or to indirectly bind the analyte by means of an ancillaryspecific binding member, which is described in greater detailhereinafter. The label reagent can be incorporated into the Test Deviceat a site upstream from the capture zone, it can be combined with thefluid sample to form a fluid solution, it can be added to the testdevice separately from the test sample, or it can be predeposited orreversibly immobilized at the capture zone. In addition, the specificbinding member may be labeled before or during the performance of theassay by means of a suitable attachment method.

“Label” refers to any substance which is capable of producing a signalthat is detectable by visual or instrumental means. Various labelssuitable for use in the present invention include labels which producesignals through either chemical or physical means. Such labels caninclude enzymes and substrates, chromogens, catalysts, fluorescent orfluorescent like compounds and/or particles, magnetic compounds and/orparticles chemiluminescent compounds and or particles, and radioactivelabels. Other suitable labels include particulate labels such ascolloidal metallic particles such as gold, colloidal non-metallicparticles such as selenium or tellurium, dyed or colored particles suchas a dyed plastic or a stained microorganism, organic polymer latexparticles and liposomes, colored beads, polymer microcapsules, sacs,erythrocytes, erythrocyte ghosts, or other vesicles containing directlyvisible substances, and the like. Typically, a visually detectable labelis used as the label component of the label reagent, thereby providingfor the direct visual or instrumental readout of the presence or amountof the analyte in the test sample without the need for additional signalproducing components at the detection sites.

Additional labels that can be utilized in the practice of the inventioninclude, chromophores, electrochemical moieties, enzymes, radioactivemoieties, phosphorescent groups, fluorescent moieties, chemiluminescentmoieties, or quantum dots, or more particularly, radiolabels,fluorophore-labels, quantum dot-labels, chromophore-labels,enzyme-labels, affinity ligand-labels, electromagnetic spin labels,heavy atom labels, probes labeled with nanoparticle light scatteringlabels or other nanoparticles, fluorescein isothiocyanate (FITC), TRITC,rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7,Texas Red, Phar-Red, allophycocyanin (APC), epitope tags such as theFLAG or HA epitope, and enzyme tags such as alkaline phosphatase,horseradish peroxidase, I²-galactosidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase and hapten conjugates such asdigoxigenin or dinitrophenyl, or members of a binding pair that arecapable of forming complexes such as streptavidin/biotin, avidin/biotinor an antigen/antibody complex including, for example, rabbit IgG andanti-rabbit IgG; fluorophores such as umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, eosin,green fluorescent protein, erythrosin, coumarin, methyl coumarin,pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue,dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin,fluorescent lanthanide complexes such as those including Europium andTerbium, Cy3, Cy5, molecular beacons and fluorescent derivativesthereof, a luminescent material such as luminol; light scattering orplasmon resonant materials such as gold or silver particles or quantumdots; or radioactive material include ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,Tc99m, ³⁵S or ³H; or a spherical shells, and probes labeled with anyother signal generating label known to those of skill in the art. Forexample, detectable molecules include but are not limited tofluorophores as well as others known in the art as described, forexample, in Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz(Editor), Plenum Pub Corp, 2nd edition (July 1999) and the 6^(th)Edition of the Molecular Probes Handbook by Richard P. Hoagland.

A number of signal producing systems may be employed to achieve theobjects of the invention. The signal producing system generates a signalthat relates to the presence of an analyte (i.e., target molecule) in asample. The signal producing system may also include all of the reagentsrequired to produce a measurable signal. Other components of the signalproducing system may be included in a developer solution and can includesubstrates, enhancers, activators, chemiluminescent compounds,cofactors, inhibitors, scavengers, metal ions, specific bindingsubstances required for binding of signal generating substances, and thelike. Other components of the signal producing system may be coenzymes,substances that react with enzymic products, other enzymes andcatalysts, and the like. In some embodiments, the signal producingsystem provides a signal detectable by external means, by use ofelectromagnetic radiation, desirably by visual examination. Exemplarysignal-producing systems are described in U.S. Pat. No. 5,508,178.

In some embodiments, nucleic acid molecules can be linked to thedetection probe (e.g., antibody-linked oligonucleotides), whereby thenucleic acid functions as a label by utilizing nucleic acid labels. Forexample, a reagent solution or substrate comprised in a SCD can comprisedetection reagents—plurality of detection and capture specific bindingagents (“SBA”)—comprising a plurality of oligonucleotides functioning toprovide a detectable signal, whereby for a given subpopulation ofSBAs(specific for a particular analyte), conjugated oligonucleotides arepre-stained with a different stain as compared to another subpopulationof antibodies (specific for a different analyte) are nucleic acid stainsthat bind nucleic acid molecules in a sequence independent manner.Examples include intercalating dyes such as phenanthridines andacridines (e.g., ethidium bromide, propidium iodide, hexidium iodide,dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, andACMA); some minor grove binders such as indoles and imidazoles (e.g.,Hoechst 33258, Hoechst 33342, Hoechst 34580 and DAPI); and miscellaneousnucleic acid stains such as acridine orange (also capable ofintercalating), 7-AAD, actinomycin D, LDS751, and hydroxystilbamidine.All of the aforementioned nucleic acid stains are commercially availablefrom suppliers such as Molecular Probes, Inc. Still other examples ofnucleic acid stains include the following dyes from Molecular Probes:cyanine dyes such as SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1,POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3,PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5,JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen,SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43,-44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15,-14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -63 (red). Other detectable markers includechemiluminescent and chromogenic molecules, optical or electron densitymarkers, etc.

As noted above in certain embodiments, labels comprise semiconductornanocrystals such as quantum dots (i.e., Qdots), described in U.S. Pat.No. 6,207,392. Qdots are commercially available from Quantum DotCorporation. The semiconductor nanocrystals useful in the practice ofthe invention include nanocrystals of Group II-VI semiconductors such asMgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS,ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe as well as mixedcompositions thereof; as well as nanocrystals of Group III-Vsemiconductors such as GaAs, InGaAs, InP, and InAs and mixedcompositions thereof. The use of Group IV semiconductors such asgermanium or silicon, or the use of organic semiconductors, may also befeasible under certain conditions. The semiconductor nanocrystals mayalso include alloys comprising two or more semiconductors selected fromthe group consisting of the above Group III-V compounds, Group II-VIcompounds, Group IV elements, and combinations of same.

In some embodiments, a fluorescent energy acceptor is linked as a labelto a detection probe (i.e., binding moiety conjugated with a detectormolecule). In one embodiment the fluorescent energy acceptor may beformed as a result of a compound that reacts with singlet oxygen to forma fluorescent compound or a compound that can react with an auxiliarycompound that is thereupon converted to a fluorescent compound. Suchauxiliary compounds can be comprised in buffers contained in an SCDand/or Test Device. In other embodiments, the fluorescent energyacceptor may be incorporated as part of a compound that also includesthe chemiluminescer. For example, the fluorescent energy acceptor mayinclude a metal chelate of a rare earth metal such as, e.g., europium,samarium, tellurium and the like. These materials are particularlyattractive because of their sharp band of luminescence. Furthermore,lanthanide labels, such as europium (III) provide for effective andprolonged signal emission and are resistant to photo bleaching, therebyallowing Test Devices containing processed/reacted sample to be setaside if necessary for a prolong period of time.

Long-lifetime fluorescent europium(III) chelate nanoparticles have beenshown to be applicable as labels in various heterogeneous andhomogeneous immunoassays. See, e.g., Huhtinen et al. Clin. Chem. 2004October; 50(10): 1935-6. Assay performance can be improved when theseintrinsically labeled nanoparticles are used in combination withtime-resolved fluorescence detection. In heterogeneous assays, thedynamic range of assays at low concentrations can be extended.Furthermore, the kinetic characteristics of assays can be improved byuse of detection antibody-coated high-specific-activity nanoparticlelabels instead of conventionally labeled detection antibodies. Inhomogeneous assays, europium(III) nanoparticles have been shown to beefficient donors in fluorescence resonance energy transfer, enablingsimple and rapid highthroughput screening. Heterogeneous and homogeneousnanoparticle-label-based assays can be run with various sample matrixes,e,g., serum, heparin plasma, and mucus.

In some embodiments, a label (e.g., fluorescent label) disclosed herein,is comprised as a nanoparticle label conjugated with biomolecules. Inother words, a nanoparticle can be utilized with a detection or captureprobe. For example, a europium(III)-labeled nanoparticle linked tomonoclonal antibodies or streptavidin (SA) to detect a particularanalyte in a sample can be utilized in practice of the present invention(e.g., nanoparticle-based immunoassay). The nanoparticles serve as asubstrate to which are attached the specific binding agents to theanalyte and either the detection (i.e., label) or capture moiety.

In various embodiments of the invention, the label utilized is alanthanide metal. Lanthanides include but are not limited to europium,samarium, terbium or dysprosium. Non-specific background fluorescencehas a decay time of only about 10 ns, so that such background dies awaybefore the sample fluorescence is measured. Furthermore,Lanthanidechelates have large Stokes' shifts. For example, the Stokes'shift for europium is almost 300 nm. This big difference betweenexcitation and emission peaks means that the fluorescence measurement ismade at a wavelength where the influence of background is minimal. Inaddition, the emission peak is very sharp which means that the detectorcan be set to very fine limits and that the emission signals fromdifferent lanthanide chelates can be easily distinguished from eachother. Therefore, in one embodiment, one or more different lanthanidescan be utilized in the same assay.

Hard Standards. In one embodiment, a fluorescence reader is configuredto comprise an integrated or permanent standard (“hard standard”). Theterm “hard standard” as referred to herein means that the device forreading a test sample in methods of detecting/quantifying one or moreanalytes comprises an internal, integrated or permanent standard,against which samples labeled with the same label as that used in thehard standard are read. In one embodiment, the hard standard and thetest label comprise a lanthanide (e.g., Europium III) FIG. 33.

In one embodiment, the reader is an LED, comprising a lamp emitting UV A(400 to 315 nm) part of the spectrum. Emission is in the visible part ofthe spectrum. Some exemplary or conventional LEDs or photodiodes aredisclosed in U.S. Pat. Nos. 7,175,086, 7,135,342, and 7,106,442, thedisclosure of each of which is incorporated herein in its entirety.

In another embodiment, a reader comprises at least two hard standards ofdifferent amounts (e.g., low and high concentration of label), thusproviding a two point check of the reader. For example, two (2)lanthanide hard standards (e.g., Europium) are mounted permanently onthe reader slides and may be read during the course of each test read.As such, the two hard standards can be utilized to determine the lowerdetection limit (i.e., in a analyte quantification assay or fordetermining lowest detection threshold in qualitative assays). Here,fluorescence is read and plotted as percentage of fluorescence (y axis)against concentration (x axis). The straight line between the two readsfor each of the hard standards on such a plot allows measuring theintercept of noise (no label) to give a measurement for the lowestdetection limit.

In some embodiments, a Test Device comprises a chamber (compartment orliquid sac) that contains wash or running buffer, which functions toremove unbound label, to reduce or eliminating background noise. Invarious embodiments, devices comprising a hard standard (s) provideaccurate qualitative as well quantitative measurement of analyte(s)present in a sample and labeled with label that is the same as that usedin the hard standard(s).

In some embodiments, hard standards are embedded or cast in a polymermaterial, including glass, plastic, vinyl, or acrylic FIG. 33B. Suchembedded labels can be cast into appropriate shapes/sizes.Alternatively, such hard standards can be cut to appropriate sizes to beintegrated into a reader. In one embodiment, hard standards are cut inrectangular, square, oblong, circular, or any polygon shape. In oneembodiment, hard standards are cut into rectangular shapes, comprisingdimensions for height of about 0.04, 0.045, 0.05, 0.055, 0.06, 0.065,0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.10, 0.11, 0.12, 0.125, 0.126,0.127, 0.128, 0.129, 0.130, 0.135, 0.140, 0.150 inch; width of about0.01, 0.02, 0.03, 0.035, 0.039, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0,1,0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75,0.8, 0.85, 0.9, 0.95, or 1.0 inch; and lengths of about 0.01, 0.02,0.03, 0.035, 0.039, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2,0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85,0.9, 0.95, or 1.0 inch.

In one embodiment, a reader employing a hard standard as a reference isutilized for normalizing readers across a population, e.g., plottingsubsequent reader performance against a pre-determined “Gold Standard”reader as illustrated in the following table:

TABLE 4 Gold Std. Test S0 1000 900 S1 5400 5000 S2 10200 11000 S3 1900020000 S4 22000 23000 S5 50000 50000

Therefore, where y and x axis are Test reader and Gold Standardmeasurements respectively, the lower limit of detection is the interceptof the plotted line across the noise level (reading with no label).

In one embodiment, a Test Device comprises different pRNAs eachpatterned based on a specific analyte, a complementary SCD comprises aplurality of capture antibody linked to cognate pRNAs to thoseimmobilized on the Test Device, and where said plurality comprisingdifferent subpopulation of antibodies specific for different analytes).Furthermore, the SCD reagent solution or substrate (e.g., lyophilizedsolid substrate) comprise detection probes, or a plurality ofeuropium(III) labeled antibodies, consisting of the same subpopulationsof antibodies specific for different analytes. Additional lanthanidelabels that can be practiced in the present invention are known in theart, such as disclosed in U.S. Pat. No. 7,101,667. See also, e.g.,Richardson F. S., “Terbium(III) and Europium(III) Ions as Luminescentprobes and Stains for Biomolecular Systems,” Chem. Rev., 82:541-552(1982).

Therefore, depending on choice of labels, in some embodiments a signalis viewable by the unaided eye, while in other embodiments, a readerinstrument is utilized in the practice of the present invention.

Capture Moieties.

In some embodiments, one member of a pair of complementary capturemoieties will be bound to analyte-specific binding agent and the othermember is immobilized on a line or spot FIG. 8A, respectively. Asreferred to herein, the terms “capture moiety” means a binding moietythat is specific for a partner or complementary capture moiety (e.g.,pRNA specific for complementary pRNA, or avidin/streptavidin-bioin). Inone embodiment, a Test Device comprises a combination of differentcapture moieties, wherein said capture moieties are comprise ofdifferent substances in number and/or type. For example, a test stripdisposed in a Test Device can comprise one or more addressable testlines comprising pRNA, antibodies and specific binding members (e.g.,avidin/biotin). Therefore, in various embodiments where two of the sametype of capture moieties are disposed on a test strip, each will bespecific for a different complementary partner molecule. For example,for two pRNA addressable lines, each line will comprise pRNA havingdifferent sequences which will specifically bind to pRNA ofcomplementary sequences which are themselves bound to antibodiestargeting a different analyte (e.g., Influenza A versus Influenza B).Furthermore, in such a configuration, additional test lines can comprisedifferent capture moieties (e.g., antibodies or avidin) which themselvesbind their cognate partner molecules. Thus in one embodiment, a teststrip comprise 2, 3, 4, 5, 6, 7 or 8 addressable test lines, which cancomprise any combination of 1, 2, 3 or 4 different types of capturemoieties.

Where multiplexed (i.e., multianalyte) detection is desired, a pluralityof capture moieties is utilized, antibodies specific for one analyte(s)will comprise a member of one specific pair of complementary capturemoieties and antibodies that specifically bind a second and differentanalyte(s) will comprise a member of a second and different specificpair of complementary capture moieties, and so on. Thus, in oneembodiment, a plurality of different analyte(s) can be detected, wherethe cognate member of a pair of capture moieties is immobilized in adiscrete location on a test membrane comprised in the test implement.For example, a plurality of antibodies in an SCD is comprised ofantibodies targeting different influenza virus strains and/or subtypes,where said antibodies are comprised of pairs of detectionantibody-capture antibody and where the capture antibody has a specificcapture moiety. Further, each population of antibodies in the pluralityof antibodies is defined by the particular target analyte to which theantibody binds. Thus, all capture antibodies directed to one specifictarget analyte will have the same capture moiety, for whichcognate/complementary capture moieties are disposed in the Test Device.

In various embodiments, capture moieties are comprised of anoligonucleotide, avidin, streptavidin, pyranosyl RNA (pRNA),antigen-antibody binding pair selected for high affinity, aptamer or acombination thereof. In further embodiments, an oligonucleotide is DNAor RNA. Moreover, in some embodiments a combination of differentcapturemoieties are utilized in the same detection system of theinvention. For example, a capture moiety pair for one specific analytecomprises an oligonucleotide pair, while a capture pair for a differentanalyte comprises a capture moiety pair comprising pRNA, or avidin orstreptavidin, etc. In other embodiments, a combination of differenttypes of capture moieties is utilized in devices and assays of theinvention to detect multiple analytes (e.g., plurality of captureantibodies whereby each population of capture antibodies specific for asingle type of target analyte is linked to one type of capture moieties,and other analyte-specific antibodies are linked to others, such asaptamers, pRNA or streptavidin, etc.)

In one embodiment, all capture moieties are pRNAs, with multiple pairsof pRNA capture moiety and pRNA partner capture moiety (e.g., one isconjugated to a specific binding agent and the cognate pRNA isimmobilized on the lateral flow membrane).

pRNA

In one aspect of the invention, combinations of complementary pyranosylRNA (pRNA) sequences are incorporated in the SCD/Test Devices of theinvention enabling simultaneous specific detection of multiple analytesFIG. 32. In various embodiments one of a pair of homologous pRNAsequences is immobilized in a specific stripe or test zone in the TestDevice, while the other of the pair of homologous pRNA sequences isconjugated to a binding moiety, which specifically binds to a targetanalyte. Thus in one embodiment, a target analyte is captured by theimmobilized pRNA through interaction of the immobilized pRNA sequencewith its binding pair pRNA. The analyte is detected by a detectormolecule conjugated to a second binding moiety 1301 also specific forthe same target analyte.

In some embodiments, pRNA binding partners are selected from but notlimited to the following pRNAs

TABLE 5 Name 4′-2′ SEQ ID NO: 102a10-3-NH2 TAGAACGAAG 98 102b10-3-NH2CTTCGTTCTA 99 119a10-1-NH2 TCAGTGGATG 100 119b10-1-NH2 CATCCACTGA 1013a10-1-NH2 GTATTGCGAG 102 3b10-1-NH2 CTCGCAATAC 103 102a8-2-NH2 AACGATTC104 102b8-2-NH2 GAATCGTT 105 119a8-1-NH2 AGTGGATG 106 119b8-1-NH2CATCCACT 107 3a8-1-NH2 GTATTGCG 108 3b8-1-NH2 CGCAATAC 109 4a8 ATGCCTTC110 4b8 GAAGGCAT 111 5a8 TGATGGAC 112 5b8 GTCCATCA 113 6a8 CAGTAGTG 1146b8 CACTACTG 115 7a8 TTCCTGAG 116 7b8 CTCAGGAA 117 8a8 GACTCTCT 118 8b8AGAGAGTC 119all oligos with 4′-C12 amino and 2′-hexanol groups

Therefore, where a plurality of capture probes (e.g., antibody linked topRNA), each capture probe is linked to a capture moiety, for which acognate capture probe is immobilized in a predetermined location on atest strip 310, 903, 904, 905, 906comprised in a Test Device FIGS. 3,26, 27. See also, FIGS. 39-42, which illustrate the substantialsensitivity of pRNA capture.

Therefore, a sequence immobilized on a test strip at a specificaddressable line will bind specifically to the complimentary pRNAconjugated with anti-analyte binding moieties (e.g., anti-virusantibody). The efficacy of such specificity is demonstrated in Example 6(e.g., FIG. 30).

In some embodiments, a Test Device incorporating one or more pRNAbinding pairs, provides sensitivity of about 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0,08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0,8, 0.9,1.0, 1.2, 1.5, 1.7, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5,7.0, 7.5, 8,0, 8.5, 9.0, 9.5, 10.0, 15, 20, 30, 40 or 50 ng/mL.

In some embodiments pRNA is attached to a membrane (i.e., test strip)utilizing a protein linker. For example, pRNA can be conjugated to ahydrophilic protein. In one embodiment, the linker protein has amolecular weight of at least from about 500, 600, 700, 800, 900, 1000,1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7500,8000, 9000, 1.0000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, 100000, 110000, 120000, 130000, 140000, 150000, 160000, 170000,180000, 190000, 200000, 225000, 250000, 300000, 350000 to about 450000.Such a linker can range in size from about s about 5 to 10, 6 to 11, 7to 12, 8 to 13, 9 to 14, 10 to 15, 11 to 16, 12 to 17, 13 to 18, 14 to19, 15 to 20, 16 to 21, 17 to 22, 18 to 23, 19 to 24, 20 to 25, 21 to26, 22 to 27, 23 to 28, 24 to 29, 25 to 30, 35, 40, 45 or 50 AA long.The linker can be a peptide or polypeptide. In one embodiment, thelinker is BSA or IgG.

In one embodiment, pRNA is coupled to a hydrophilic protein/peptide viaa covalent bond between the pRNA molecule and the hydrophilic protein. Asolution containing the pRNA-protein complex is applied to definedregions on a test membrane (e.g., nitrocellulose), whereby the proteinanchor binds to the membrane in a an irreversible. The pRNA is thenavailable for use in the assay. In one embodiment, the anchor/linkerprotein is a hydrophilic protein and the test membrane isnitrocellulose.

As indicated previously, a Test Device can comprise addressable testlines utilizing different types of capture moieties (e.g., a combinationof antibodies, nucleic acids, pRNA, avidin/streptavidin/biotin). In oneembodiment (e.g., FIG. 32), at least one addressable line or specificcapture zone 1305, 1312 comprises a pRNA 1304, 1311 sequence that isbound to a solid support 1308 (e.g., nitrocellulose, polystyrene, glass,plastic, metal, etc.) and is specific in binding for a homologous pRNAsequence conjugated to a second binding moiety 1302, 1310 that isspecific for a particular target analyte 1306. Furthermore, a firstbinder molecule is a partner to the first binder molecule and saidsecond binder molecule is conjugated to a detector molecule 1301, 1309(e.g., fluorescent label). As demonstrated in Example 6 herein, pRNA iseffectively immobilized and can specifically bind a complementarysequence to allow detection of an analyte. As such, pRNA provides anovel tool for detecting multiple analytes based on the greatspecificity and avidity of pRNA molecules.

In one embodiment, pRNA molecules are disposed on 1, 2, 3, 4, 5, 6, or 7distinct addressable lines (i.e., capture zones) in a Test Device. Inother embodiments, pRNA are utilized in combination with antibodies,nucleic acid binding pairs, and avidin/streptavidin,digoxin/anti-digoxin. For example, a Test Device comprise a test stripwith 5 addressable test/capture zones, wherein each test zone isspecific for a distinct analyte (e.g., influenza type A or B) and/orsubtype (e.g., influenza A pandemic and non-pandemic subtypes). Thus,for example, two test zones utilized pRNA binding, while one utilizesstrepatvidin/avidin-biotin, while another utilizes fixed antibody, andyet another utilizes DNA/RNA. In this example, for each type of bindingsystem, there is a complementary binding partner that is specific forthe target analyte which is “captured” on the particular test zone.Further, as described herein, the analyte is also bound by a binder thatis conjugated to a detector molecule/label (e.g., fluorescence label).As such, the once the analyte-detector-binding partner complex flowsthrough the Test Device, the complex is captured at the distinct testzone having the immobilized binding partner.

As such a central aspect of the present SCD/Test Devices of theinvention is that they can be configured to detect multiple analytes,including cells, cell components (e.g., cell markers, cell surfacemarkers), proteins (e.g., enzymes) and the such.

In one embodiment, SCD/Test Devices of the invention can be used in amethod to assay for any pathogenic conditions for which particularcorresponding analytes are known or are identified in future. The SCDand Test Device can be configured to provide any combination of thedetection reagents disclosed herein (e.g., pRNA, nucleic acids,antibodies, specific binding partners, e.g., avidin/biotin). Forexample, multiple analytes corresponding to myocardial infarction (MI)can be identified in detecting/diagnosing MI. Markers for variousconditions are known in the art, such as for cardiac markers disclosedin U.S. Pat. Nos. 5,604,105; 5,710,008; 5,747,274, 5,744,358 and5,290,678, the disclosures of each of which is incorporated by referenceherein in its entirety.

In one embodiment, the devices of the invention provide a three in oneassay for protein based cardiac markers of myocardial infarction wouldbe designed (See Example 10).

In one embodiment, a sample is applied in a way that enables mixing witha mixture (in series or in parallel) of a first binder conjugated to adetector reagent (e.g., combinations of gold microparticles andfluorescent label) and with a second binder conjugated to a first memberof a pRNA homologous binding pair which may or may not contain materialsto enable an immune binding reaction and/or specific binding of pRNAhomologous binding pairs.

If desired a pre-incubation is designed into the system whereby thefirst and second binders are allowed to attach to and bridge the analyteforming complex, and such a step can be during the flow of reagents tothe test or capture zone. Alternatively, the flow may be stopped toallow for the reaction to come closer to completion. In yet anotherembodiment, the reagent mix in an SCD of the invention prior to beapplied to a Test Device.

A mixture flows via capillary action or hydrostatic pressure from any ofseveral mechanism or other non-capillary action along the surface of orwithin a matrix of a solid material/substrate (e.g., test strip)allowing the first and second homologous binding pairs to come intocontact as the reaction mixture passes over/through the test/capturezone. If the first and second binder have bridged the analyte FIG. 32the detector reagent (e.g., Europium) will accumulate at thetest/capture zone yielding a signal that can be interpreted visually orusing an instrument reader.

In embodiments where multiple different analytes are sought to bedetected, different detector reagents are used for each analyte and eachdifferent analyte is captured in a single detection/capture zone. Aninstrument reader is used to distinguish between the signal for eachanalyte. In one embodiment, the instrument reader can detect a single toprovide for qualitative and quantitative measurement.

In another embodiment, unique pRNA homologous binding pairs are utilizedfor each target analyte.

In another embodiment a wash buffer or reagent buffer is releasedthrough the flow path (as described herein) where said buffer cancomprise a reagent that yields the release of energy in any of severalforms that can be detected with an instrument, reader or visually. Forexample, the reaction can result in release of light, electrons or canrequire the input of electrons plus a substrate yielding the release oflight. Such reactions are known to one of skill in the art.

Such reactions can result in enablement of a fluorescent compound tofluoresce after coming into contact with the running/wash buffer,resulting in release of electrons, protons, neutrons, which can bedetected by an instrument or reader as described herein.

Aptamers.

In some embodiments, capture moieties are aptamer molecules that are canbe interchangeably utilized with a capture probe or as an immobilizedcapture moiety included in the Test Device axial flow membrane. Aptamersinclude nucleic acids that are identified from a candidate mixture ofnucleic acids. In a preferred embodiment, aptamers include nucleic acidsequences that are substantially homologous to the nucleic acid ligandsisolated by the SELEX method. Substantially homologous is meant a degreeof primary sequence homology in excess of 70%, most preferably in excessof 80%. The “SELEX” methodology, as used herein, involves thecombination of selected nucleic acid ligands, which interact with atarget analyte in a desired action, for example binding to a protein,with amplification of those selected nucleic acids. Optional iterativecycling of the selection/amplification steps allows selection of one ora small number of nucleic acids, which interact most strongly with thetarget antigen/biomarker from a pool, which contains a very large numberof nucleic acids. Cycling of the selection/amplification procedure iscontinued until a selected goal is achieved. The SELEX methodology isdescribed in the following U.S. patents and patent applications: U.S.patent application Ser. No. 07/536,428 and U.S. Pat Nos. 5,475,096 and5,270,163.

Infectious Agents.

In various embodiments of the present compositions and methods, aninfectious agent can be any pathogen including without any limitationbacteria, yeast, fungi, virus, eukaryotic parasites, etc. In someembodiments, the infectious agent is influenza virus, parainfluenzavirus, adenovirus, rhinovirus, coronavirus, hepatitis viruses A, B, C,D, E, etc, HIV, enterovirus, papillomavirus, coxsackievirus, herpessimplex virus, or Epstein-Barr virus. In other embodiments, theinfectious agent is Mycobacterium, Streptococcus, Salmonella, Shigella,Staphylcococcus, Neisseria, Clostridium, or E. coli. It will be apparentto one of skill in the art that the compositions and methods of theinvention are readily adaptable to different infectious agents, byutilizing a different panel of binding agents (e.g., antibodies) thatare specific for type(s) or subtype(s) of an infectious agent(s).

Usually the general type of an infectious agent can be the genus type ofan infectious agent or any primary or first instance typing oridentification of an infectious agent. A subtype of an infectious agentcan be the species or strain type of an infectious agent or anysecondary or subsequent typing of an infectious agent. According to thepresent invention, identification of the general type or subtype of aninfectious agent can be carried out via various suitable test set ups.For example, identification of the general type of an infectious agentcan include one or more screening tests for 1) a specific general typeof an infectious agent, 2) certain desired or selected general types ofan infectious agent, or 3) all or substantially all relevant generaltypes of an infectious agent, or a combination thereof. Similarlyidentification of the subtype of an infectious agent can include one ormore screening tests for 1) one or more specific subtypes of aninfectious agent, 2) one or more specific subtypes of a particulargeneral type of an infectious agent, 3) one or more specific subtypes ofan infectious agent selected based on additional information associatedwith the subject being tested, e.g., one or more suspected or expectedsubtypes for a particular population or geographic location or 4) one ormore potentially pandemic or epidemic subtypes of an infectious agentthat is identical to or associated with the infectious agent tested forthe general type, or a combination thereof.

According to another aspect of the present invention, the methodprovided by the present invention can optionally or additionally includeidentification of the general and/or subtype(s) of a second infectiousagent that is closely related to the first infectious agent, oralternatively the infection of the second infectious agent is associatedor likely coupled with the infection of the first infectious agent. Inone embodiment, the method provided by the present invention includesidentification of the general and subtype(s) of a virus as well as abacterium. For example, HIV infection can be associated with certainbacterial infection therefore it will be useful to identify the generaland subtyp(s) of HIV as well as Mycobacterium and/or Pneumocystiscarina. Specifically the method provided by the present inventionincludes identification of HIV and one or more species of Mycobacteriumand/or Pneumocystis carina.

In another embodiment, the method provided by the present inventionincludes identification of the general and subtype(s) of a first virusas well as a second virus. For example, the method provided by thepresent invention can include identification of the general andsubtype(s) of HIV as well as hepatitis virus or alternatively thegeneral and subtype(s) of HIV as well as the general type of hepatitisvirus or certain strains of hepatitis virus. In one embodiment, themethods and compositions of the invention can be utilized in assays todetect E. coli 0157 (a very dangerous, often fatal infectious strain) inthe presence of other enteric or infective strains. Another examplewould be in testing patients for influenza infection, where mutation orvariation of the strains within subtypes is known to occur and someforms of influenza are far more pathogenic than others. A furtherexample is detection of different types of HIV, for example HIV-1 andHIV-2.In one aspect, identification of the general type of humanimmunodeficiency virus (HIV) can include screening for the presence ofHIV whereas identification of the subtype of HIV can include screeningfor HIV-1, HIV-2, and/or other subtypes of HIV. Similarly identificationof the general type of herpes virus such as simplex virus (HSV) caninclude screening for the presence of HSV whereas identification of thesubtype of HSV can include screening for HSV type 1 and/or HSV type 2 orfor Epstein-Barr virus and subtypes of EBV.

In still another particular aspect, identification of the general typeof enterovirus can include screening for the presence of one or moreenteroviruses, e.g., poliovirus, coxsackievirus, echovirus, designatedenterovirus, etc. whereas identification of the subtype of enteroviruscan include screening for poliovirus, e.g., serotype 1-3, coxsackievirusA, e.g., serotype 1-22 and 24, coxsackievirus B, e.g., serotype 1-6,echovirus, e.g., serotype 1-9, 11-27, 29-31, and designated enterovirus,e.g., enterovirus 68-71, etc.

In general, with respect to a bacterial infectious agent identificationof the general and subtype of a bacterial infectious agent includesscreening for the genus and one or more species or strains of thebacterial infectious agent that are relevant to the infection and/or theagent's antimicrobial resistance. In one embodiment, identification ofthe general and subtype of a bacterial infectious agent includesscreening for Mycobacterium and one or more species of Mycobacteriumincluding without limitation tuberculosis, avium, bovis, chelonei,fortuitum, intracellulare, kansasii, leprae, etc. In another embodiment,identification of the general and subtype of a bacterial infectiousagent includes screening for Salmonella and one or more species ofSalmonella including without limitation typhi, enteritidis, etc. In yetanother embodiment, identification of the general and subtype of abacterial infectious agent includes screening for Shigella and one ormore species of Shigella including without limitation dysenteriae. Inyet another embodiment, identification of the general and subtype of abacterial infectious agent includes screening for Streptococcus and oneor more species of Streptococcus including without limitation pneumonia,pyogenes (group A), etc. In still yet another embodiment, identificationof the general and subtype of a bacterial infectious agent includesscreening for E coli and one or more strains of E coli including withoutlimitation enterotoxigenic strains.

According to the present invention, screening test(s) used for theidentification of the general and subtype(s) of an infectious agent canbe any suitable tests known or later discovered in the field. Forexample, the screening tests of the present invention can be anon-nucleic acid based test including without any limitation a protein,peptide, amino acid, ligand, or chemistry based test. In one embodiment,the screening test of the present invention is a test based on thepresence or absence of one or more structural proteins of an infectiousagent, e.g., glycoproteins, envelop proteins, polysaccharides, etc. Inanother embodiment, the screening test of the present invention is atest based on the presence or absence of one or more antigens orepitopes, or antibodies to an infectious agent. In yet anotherembodiment, the screening test of the present invention is a test basedon the presence or absence of one or more substances that is released ormetabolized by an infectious agent. In still yet another embodiment, thescreening test of the present invention is a test based on the presenceor absence of one or more substances derived from a host cell associatedwith or generated by the infection of an infectious agent,

In one particular aspect, the non-nucleic acid based screening test ofthe present invention includes the commonly used ligand binding orimmunoassays, e.g., ligand or immunochromatographic assays. Many ofthese assays are based on the highly specific interactions betweenspecific binding pairs. Examples of such binding pairs includeantigen/antibody, hapten/antibody, lectin/carbohydrate,apoprotein/cofactor and biotin/(strept)avidin. Furthermore, many ofthese assays involve devices (e.g., solid phase, lateral-flow teststrips, flowthrough tests) with one or more of the members of a bindingpair attached to a mobile or immobile solid phase material such as latexbeads, glass fibers, glass beads, cellulose strips or nitrocellulosemembranes (U.S. Pat. Nos. 4,703,017; 4,743,560; 5,073,484).

In one embodiment, the methods and apparatus of the invention areutilized to detect or identify an influenza type A subtype and/orinfluenza type B and/or influenza type C.

Influenza virus belongs to the genus orthomyxovirus in the family ofOrthomyxoviridae. ssRNA enveloped viruses with a helical symmetry.Enveloped particles 80-120 nm in diameter. The RNA is closely associatedwith the nucleoprotein (NP) to form a helical structure. The genome issegmented, with 8 RNA fragments (7 for influenza C). There are 4principle antigens present, the hemagglutinin (H), neuraminidase (N),nucleoprotein (NP), and the matrix (M) proteins. The NP is atype-specific antigen which occurs in 3 forms, A, B and C, whichprovides the basis for the classification of human and non-humaninfluenza viruses. The matrix protein (M protein) surrounds thenucleocapsid and makes up 35-45% of the particle mass. Furthermore, 2surface glycoproteins are seen on the surface as rod-shaped projections.The haemagglutinin (H) is made up of 2 subunits, H1 and H2.Haemagglutinin mediates the attachment of the virus to the cellularreceptor. Neuraminidase molecules are present in lesser quantities inthe envelope. The antigenic differences of the hemagglutinin and theneuraminidase antigens of influenza A viruses provide the basis of theirclassification into subtypes. e.g., A/Hong Kong/1/68 (H3N2) signifies aninfluenza A virus isolated from a patient in 1968, and of subtype H3N2.

In various embodiments, the methods and apparatus of the invention aredirected to detecting or identifying influenza virus type A which isdefined by HxNy where x is 1-16 and y is 1-9, or any combination of xythereof. For example, in one embodiment, the methods and apparatus ofthe invention is utilized to detect influenza A subtype H1N5. Thus, aplurality of detection probes and capture probes targeting differentsubtypes of influenza virus are disposed in an SCD of the invention. Inone embodiment, the assay is utilized to detect Influenza A (withsubtypes H1/H3, and a pandemic subtype H5) and Influenza B.

In particular, the general type of an influenza virus can be any typedesignated based on antigenic characteristics of the nucleoprotein andmatrix protein antigens, e.g., type A, B, or C influenza virus, whereasthe subtype can be one or more subdivided types of an influenza virus onthe basis of an antigen, e.g. one or more subtypes of influenza type Aor type B virus characterized by a surface antigen such as hemagglutinin(H) or neuraminidase (N).

In one embodiment, identification of the general type of influenza virusincludes screening for type A, type B, and/or type C influenza viruswhereas identification of the subtype of influenza virus, e.g., type Avirus includes screening for one or more expected subtypes of type A,e.g., subtypes expected to be present in the population at the time oftesting, and optionally one or more suspected subtypes, e.g., subtypesunder surveillance for an outbreak such as epidemic or pandemicoutbreak. In another embodiment, identification of the general type ofinfluenza virus includes screening for type A and type B influenza viruswhereas identification of the subtype of influenza virus, e.g., type Avirus includes screening for one or more subtypes used for theproduction of the influenza vaccine, e.g., current vaccine subtypes(s)or strain(s) for the testing season including subtypes and/or strainsexpected to be in circulation during the next influenza season. In yetanother embodiment, identification of the general type of influenzavirus includes screening for type A and type B influenza virus whereasidentification of the subtype of influenza virus, e.g., type A includesscreening for one or more subtype(s) or strain(s) used for theproduction of the influenza vaccine and one or more subtype(s) orstrain(s) suspected for the cause of a pandemic outbreak, e.g., one ormore avian subtype(s) or strain(s) such as H5N1 or the derivativesthereof.

In yet another embodiment, identification of general type of influenzavirus includes screening for type A and type B influenza virus whereasidentification of the subtype of an influenza virus, e.g., type Aincludes screening for one or more common or expected subtypes incirculation including, without any limitation, a) H₁ and H₃, b) H₁, H₃,and H2, c) H₁, H₂, H₂, H₃, and H₉, d) H₁, H₃, and N₁, e) H₁, H₃, N₁, andN₂, f) H₁, H₃, and N₂, g) N₂, and h) N₁ and N₂. For example, a screeningtest for the subtype identification of type A influenza virus can bedirected to the identification of the presence of any one of thesubtypes listed in the subtype group of a), b), c), d), e), f), g), orh) e.g., without necessarily identifying the presence of a specificsubtype in a subtype group. Alternatively screening test for the subtypeidentification of type A influenza virus can be directed to theidentification of the presence or absence of each and everyone of thesubtypes listed in a), b), c), d), e), f), g), or h) e.g., identifyingthe presence of a specific subtype in a subtype group.

In still another embodiment, identification of general type of influenzavirus includes screening for type A and type B influenza virus whereasidentification of the subtype of an influenza virus, e.g., type Aincludes screening for one or more pandemic or un-expected subtypes incirculation including, without any limitation, a) H₅, b) H₅ and H₇, c)H₅, H₇, and H₉, d) N₂, N₇, and N₈, e) H₅ and N₂, f) H₅ and N₁, g) H₅ andN₈, h) H₅, N₈, H₇, and N₇, i) H₅, H₇, H₉, N₇, and N₈. For example, ascreening test for the subtype identification of type A influenza viruscan be directed to the identification of the presence of any one of thesubtypes listed in the subtype group of a), b), c), d), e), f), g), h),or i) e.g., without necessarily identifying the presence of a specificsubtype in a subtype group. Alternatively screening test for the subtypeidentification of type A influenza virus can be directed to theidentification of the presence or absence of each and everyone of thesubtypes listed in a), b), c), d), e), f), g), h), or i), e.g.,identifying the presence of a specific subtype in a subtype group.

In another particular aspect, the general type of hepatitis virus can beA, B, and C virus with each virus possibly having several subtypesincluding mutant strains. In one embodiment, identification of thegeneral type of hepatitis virus includes screening for A, B, and/or Chepatitis virus whereas identification of the subtype of hepatitis virusincludes screening for subtypes or mutant strains of A, B, and Chepatitis viruses, respectively. In another embodiment, identificationof the general type of hepatitis virus includes screening for hepatitisB virus whereas identification of the subtype of hepatitis virusincludes screening for one or more subtypes and/or mutant strains ofhepatitis B virus. In yet another embodiment, identification of thegeneral type of hepatitis virus includes screening for hepatitis C viruswhereas identification of the subtype of hepatitis virus includesscreening for one or more of subtypes 1-9 of type C hepatitis virus.

In various embodiments, methods and apparatus of the invention candetect one or more different infectious agents. For example, a samplingimplement can comprise a plurality of different antibodies, whereinmultiple subgroups of antibodies are present, whereby each subgroup ofantibodies specifically binds a different infectious agent. For example,a plurality of antibodies can comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10subgroups, wherein each subgroup of antibodies in the plurality ofantibodies specifically binds a different infectious agent. In someembodiments, methods and apparatus of the invention detect a pandemicand non-pandemic infectious agent. In one embodiment, the pandemic andnon-pandemic infectious agents are influenza virus.

The explosive nature of epidemic influenza and the specific clinicalfeatures of this disease have given reliable epidemiological records ofthis infection since the beginning of the nineteenth century. Severalepidemics were recorded during the nineteenth century but the firstpandemic was not accurately recorded until 1889-92. A second pandemic,probably originating in Europe, occurred in 1918-19, and is known asSpanish Influenza, which was responsible for 20-25 million deaths,principally in young adults.

Pandemics continued to occur regularly after the Spanish influenza, in1932-33, 1947-48, 1957 and 1968. The next pandemic is thought to beoverdue. These latter pandemics resembled the pandemic of 1890,affecting millions of people with a mild URTI and a small number ofdeaths. The H1N1 (swine) viruses probably appeared in 1918 and continuedto circulate until 1957, at which time they were supplanted by the H2N2(Asian) viruses. The H2N2 viruses were prevalent until 1968, when H3N2(Hong Kong) strains appeared. The H1N1 virus reappeared in 1977 and didnot replace the H3N2 subtype and both subtypes continued to cocirculate.Therefore, it is imperative that subjects are screened in an effectiveand accurate manner to determine with what strain and/or subtype anindividual is infected. Furthermore, in some circumstances such samplecollection and processing will necessarily occur in a point-of-caresetting (e.g., in the field, without large numbers of subjects to sampleand process, and with limited man power to effect such sampling).

As such, in one embodiment, the methods and apparatus of the inventionare utilized in processing a large number of samples, in a point-of-caresetting, where test results may be visualized (i.e., read) some periodof time after the test is complete. For example, the period of time canbe 30 minutes, 1 hour, 1.5 hour, 2 hours, 2.5 hours, 3 hours, 4 hours or5 hours. In some embodiments, methods and apparatus in conjunction withthe reagents disclosed herein provide high sensitivity and specificitywhere the fluorescent result can be read with very similar results overa long period of time. Thus, in sonic embodiments biological samples canbe collected and processed, but set aside to be read a significant timelater, which is greatly advantageous in point-of-care settings or wherea large number of samples are collected with limited manpower or time tofurther process samples.

In yet another aspect of the invention, the compositions and methods ofthe invention are directed to detecting any one or more analytes presentin a sample. As indicated above, for example, by utilizing differentbinding moieties that specifically bind markers associated with acondition, one or more analytes associated with MI can be detected.Therefore, an SCD and Test Device can comprise the necessary reagents todiagnose a disease or pathological condition, other than infectiousdiseases.

In some embodiments, the one or more analytes are markers associatedwith a pathological condition or disease. In another embodiment, the oneor more analytes are polypeptides associated with a nutrional state orcondition. In yet other embodiments, the one or more analytes are cellmarkers associated with cell cycle and growth. In another embodiment,the one or more analytes are associated with cell proliferation anddifferentiation. In one embodiment, cell markers are associated withcancer.

EXAMPLES Example 1 Assay Volume and Chasing Buffer

To explore the optimal assay sample volume that will give the bestsensitivity and to investigate if a chasing buffer will also increasethe sensitivity and performance of the test strip.

Materials: Nitrocellulose Membrane: Millipore HiFlow 135 membrane, 2.5cm in width. Cat. No. SHF1350425, Lot No. R68N46849, Code No. RK04414,roll No. 040L1. Membrane was striped with monoclonal anti-H5N1 antibody,clone 3C8 at 1.0 mg/ml. Control line was striped with 1.5 mg/m1 rabbitanti-mouse antibody. Wicking pad: 0.05% Tween-20 treated polyester pad.Alhstrom grade 6613. Prepared Absorbent pad: Ahlstrom Grade 222 paper,3.5 cm in width, purchased from Fisher, Cat. #2228-1212, lot #6150502.Anti-H5 3G4 gold conjugate was prepared by Nanogen POC at Toronto,Canada, OD of 3G4 gold conjugate at maximal absorbent peak was 112.54.Extraction buffer: 50 mM Tris-Cl, pH 8.15, 0.75 M NaCl, 1% BSA, 0.1%plutonic F68, 0.05% digested casein, 2 mM TCEP and 0.02% NaN₃,Recombinant H5 hemagglutinin (0.2 mg/mL) was provided by Nanogen POC,Toronto, Canada

Protocol: Preparation of lateral flow test strip. Nitrocellulosemembrane striped with antibodies was laminated on plastic card with 1-2mm overlap with wicking pad and absorbent pad. No cover tape was appliedon any components. Strips were cut in 5 mm in width.

Test: Extraction buffer ranged from 50-100 μL was mixed with 1.1 μL 3G4gold conjugate and with or without 2 μL recombinant H5 hemagglutinin.Test was started by inserting strip into extraction buffer and resultwas scanned at 15 min, 20 min, 25 min and 30 min. For experiment withchasing buffer, see result for detail.

Result: FIG. 9 Test the strips with different volume of ExtractionBuffer, at 15, 20, 25 and 30 minutes. FIG. 9ATest strips were tested indifferent volume of Extraction Buffer. At the 15, 20, 25 and 30 min,strips were taken out and scanned with Bio-Rad GS-800 CalibratedDensitometer. Lane 1, 2: 50 μL Extraction Buffer; Lane 3, 4: 60 μLExtraction Buffer; Lane 5, 6: 70 μL Extraction Buffer; Lane 7, 8: 80 μLExtraction Buffer; Lane 9, 10: 100 μL Extraction Buffer; FIG. 9B Comparethe test line intensity of test strip with different volume ofExtraction Buffer at different reading time.

TABLE 6 Data analysis of FIG. 9 with Quantity One. Lane Extraction PeakIntensity Number Buffer (μl) 15 min 20 min 25 min 30 min 1 50 55.6 62.363.1 62.5 2 51.1 57.3 55.4 62.6 3 60 53.5 65.7 68.5 68.9 4 51.6 63.866.2 66.5 5 70 45.6 57.9 60.4 60.7 6 42.1 57.0 61.7 63.9 7 80 39.6 53.759.8 61.5 8 42.9 57.9 64.7 65.4 9 100 35.2 49.2 54.6 55.6 10 30.8 44.949.3 49.8

FIG. 10A Test Strip #3 with various amount of extraction buffer andeffect of background clean-up on their negative control result.

TABLE 7 Data analysis of FIG. 10 with software Quantity One LaneExtraction Peak Intensity Number Buffer (μl) 15 min 20 min 1 50 3.8 5.42 4.1 4.6 3 60 3.4 4.8 4 3.4 5.5 5 70 3.4 2.9 6 2.7 4.1To various amount of extraction buffer, 1.1 μL 3G4 gold conjugate wasadded. Test was started by inserting strip to the assay solution andscanned at 15 min. (First roll). Then all the strips were transferred totubes containing extraction buffer only and ran for 5 more minutes. Thestrips were scanned again. (Second roll).

FIG. 10B Test Strip #3 in various amount of extraction buffer and effectof background clean-up on their sensitivity.

To various amount of Extraction Buffer, 1.1 μL 3G4 gold conjugate and 2μL recombinant H5 hemagglutinin was added. Test was started by insertingstrip to the assay solution and scanned at 15 min. (First roll). Thenall the strips were transferred to tubes containing extraction bufferonly and ran for 5 more minutes. The strips were scanned again. (Secondroll).

TABLE 8 Data analysis of FIG. 4 with software Quantity One LaneExtraction Peak Intensity Intensity Number Buffer (μL) 15 min 20 minchange 1 50 56.9 88.7 56% 2 57.8 94.4 63% 3 60 50.2 78.3 56% 4 51.2 77.451% 5 70 46.8 67.1 43% 6 47.9 73.4 53%

Discussion. In the design of most of the rapid lateral flow tests forpoint-of-care, antibody conjugate usually is dried on a conjugate padwhich is interfaced with nitrocellulose membrane. The disadvantage ofthis design is that dried conjugate may not release evenly andconsistently from strip to strip, and from time to time due to materialsfor conjugate pad. Conversely, if antibody conjugate is premixed withassay solution, as long as the liquid flow is consistent, which is morelikely to occur because once the wicking pad is wet, the flow speedactually is controlled by the membrane rather than the wicking material,the flow of antibody conjugate should be more consistent and even.Therefore in the above example, antibody gold conjugate was directlymixed with assay solution.

Initially, 100 μL Extraction Buffer was used for each test and thereading time of the assay was set at 15 min. It was found that at theend of 15 min, not all assay solutions were consumed. There was a wasteof both analyte and gold conjugate. Experiment was designed toinvestigate what the optimal assay volume is in a relatively shortreading time ranges. FIG. 9B and table 2 showed that with larger volume,such as 100 μL, of assay solution, longer time is required to get bettersensitivity. However, with 50 μL of assay buffer, the color intensity ofthe test line does not increase much over different reading time after15 min indicating that most of the analyte and gold conjugate havepassed the capture line at 15 min. Although at 30 min the colorintensity of the stripes tested with 100 μL assay solution almost reachthe same level as those tested with low volume assay solution at 15 min,it is obvious that a 15 min test is more competitive than a 30 min testin the market.

However, due to the higher concentration of gold conjugate in arelatively small volume of assay solution, the background of test stripsshowed significant red background which reduced the contrast between thetest line and the background and was cosmetically unattractive. FIG. 10Bshowed that running a chasing buffer after 15 min could significantlyclean up the background; hence increase the sensitivity of the test by43%-63%. The data also demonstrated again that a small assay volume, 50μL, is a better choice for the test.

Example 2 Europium Bead Versus Prior Art System

Materials: Inactivated influenza A virus: Texas 1/77 (H3N2) fromMicrobix Biosystems, Inc. Cat. #EL-13-02, lot #13037A3; Inactivatedinfluenza B virus: Hong Kong 5/72 from Microbix Biosystems, Inc. Cat.#EL-14-03, lot #14057A2; Quidel QuickVue A+B Test, lot #702391

Protocol: Virus dilution-Influenza A and influenza B viral preparationswere diluted with saline to 4096 HA/mL and 409.6 HA/mL, respectively,before use. Assay procedure: Procedures described in the package insertof the Quidel test kit were followed and briefly reviewed here. Dispenseall of the Extraction Reagent Solution from the reagent tube. Gentlyswirl the tube to dissolve its content. Spike virus into the tube. Placethe swab into the Extraction Tube. Roll the swab at least three timeswhile pressing the head against the bottom and side of the ExtractionTube. Leave the swab in the tube for 1 min. Roll the swab head againstthe inside of the tube as you remove it. Place the test strip into theExtraction Tube. Read result at 10 min.

Result: FIG. 11 Test QuickVue Flu A+B with different amount of influenzaA virus, Texas 1/77. The test line is shown above the control line.

TABLE 9 Visual read result and data analysis of FIG. 4 with softwareQuantity One. Flu A virus (HA/test) Lane No. Peak intensity Visual readresult 0 1 2.5 − 2 1.9 − 1.0 3 3.1 − 4 3.1 − 2.0 5 3.0 − 6 3.9 − 4.1 77.2 + 8 8.7 + 8.2 9 18.2 + 10 14.3 + 16.4 11 21.1 + 12 31.9 + 32.8 1366.2 + 14 48.1 + 65.5 15 56.8 + 16 74.0 +

Peak intensity was analyzed with software Quantity One. Values weregiven by the software. Any values below 4 will be read as a negativewith eyes.

FIG. 12 Test QuickVue Flu A+13 with different amount of influenza Bvirus, Hong Kong 5/72.

The test line for Type B is below the control line.

TABLE 10 Visual read result and data analysis of FIG. 4 with softwareQuantity One. Flu B virus (HA/test) Lane No. Peak Intensity Visual readresult 0 1 2.9 − 2 0.2 − 0.41 3 1.6 − 4 1.9 − 0.82 5 2.3 − 6 3.5 − 1.647 4.2 ± 8 6.3 + 3.28 9 2.0 − 10 8.1 + 6.55 11 16.3 + 12 14.0 +

Peak intensity was analyzed with software Quantity One. Values weregiven by the software. Any values below 4 will be yield a negativeresult with a visual read.

TABLE 11 Test Quidel QuickVue Flu A + B with different amount ofinfluenza B virus, Hong Kong 5/72 Virus 0 0.41 0.82 1.64 (HA/test) No. 12 1 2 1 2 1 2 Visual − − ± ± ± ± + + Result

Example 3 Test the Detection Limit of Influenza A and B Test Using GoldLabel

Materials: Anti-Flu A M4090913 gold conjugate was prepared by NanogenPOC at Toronto, Canada. OD was 102.69 at maximal absorbent peak.Anti-Flu B antibody 2/3 gold conjugate was prepared by Nanogen POC atToronto, Canada. OD was 94.78 at maximal absorbent peak. See above fornitrocellulose membrane, wicking pad and absorbent pad. Membrane stripedwith anti-Flu B M2110171 antibody at 1.5 mg/ml and rabbit anti-mouseantibody at 1.5 mg/ml was prepared by Nanogen POC at Toronto, Canada.Membrane striped with anti-Flu A 7304 antibody at 1.5 mg/ml and rabbitanti-mouse antibody at 1.5 mg/ml was prepared by Nanogen POC at Toronto,Canada. Inactivated influenza A H3N2, Texas 1/77 was purchased fromMicrobix Biosystems, Inc. lot #13037A3, 40960 HA/mL.

Inactivated influenza B, Hong Kong 5/72 was purchased from Microbix,Biosystems, Inc., lot #14057A2, 40960 HA/mL; Molecular Biology Water,lot #318105; 3× extraction buffer containing 0.15 M Tris-Cl, pH 8.0,2.25 M NaCl, 0.3% Pluronic F68, 3% BSA, 6 mM TCEP, 1.5% digested caseinand 0.06% NaN₃; 2× extraction buffer containing 0.1 M Tris-Cl, pH 8.0,1.5 M NaCl, 0.2% Pluronic F68, 2% BSA, 4 mM TCEP, 1% digested casein and0.04% NaN₃; 1× extraction buffer, lot #2232-003.

Protocol: Preparation of lateral flow test strip (See above); Virusdilution: Inactivated influenza A, Texas 1/77 was diluted to 409.6 HA/mLwith 10 mM PBS with 1% BSA. Inactivated influenza B, Hong Kong 5/72 wasdiluted to 40.96 HA/mL with the same buffer before use. Influenza A andB test with gold conjugate: For influenza A test, 16.7 μt 3× extractionbuffer, 1.95 μL M4090913 gold conjugate and different amount ofinactivated influenza A virus were added to a tube. H₂O was added to afinal volume of 50 μL. For influenza B test, 25 μL 2× extraction buffer,2.12 μL 2/3 gold conjugate and different amount of virus were added to atube. H₂O was added to a final volume of 50 μL. Test was started byinserting the test strip into the assay solution. At 15 min, 50 μL 1×Extraction Buffer was added to the same tube and test was carried on for5 more minutes. Results on strips were read with eyes and also analyzedwith software Quantity One after strips were scanned with Bio-Rad GS-800Calibrated desitometer.

Result: FIG. 13. Test the limit of detection of influenza A test usinggold as label.

TABLE 12 Data analysis of FIG. 7 with software Quantity One and visualread result Virus Peak Peak Visual HA/test Lane No. Intensity Intensity(avg) read 0 1 0 0 − 2 0 0.105 3 2.2 2.8 − 4 3.3 0.21 5 4.8 6.7 + 6 8.60.41 7 13.2 11.2 + 8 9.1 0.82 9 14.8 14.1 + 10 13.5 1.64 11 26.4 24.5 +12 22.5

Values of peak intensity are the analysis result with Quantity One.

FIG. 14 Test the limit of detection of influenza B test using gold aslabel.

TABLE 13 Data analysis of FIG. 9with software Quantity One and visualread result Virus Peak Peak Visual HA/test Lane No. Intensity Intensity(avg) read 0 1 2.2 2.18 − 2 2.2 0.021 3 2.8 2.78 − 4 2.7 0.041 5 4.84.67 ± 6 4.6 0.082 7 7.3 7.76 + 8 8.2 0.164 9 14.4 13.83 + 10 13.3

Values of peak intensity are the analysis result with Quantity One.

Example 4 Test the Detection Limit of Influenza A and B Test UsingFluorescent Europium Conjugate

Materials: Wicking pad: 0.05% tween-20 treated polyester pad, 1.4 cm inwidth provided by Nanogen POC in Toronto, Canada; Absorbent pad:Ahlstrom Grade 222 paper, 3.5 cm in width, purchased from Fisher, Cat.#2228-1212, lot #6150502; Nitrocellulose Membrane: Millipore HiFlow 135membrane, 2.5 cm in width. Cat. No. SHF 1350425, Lot No. R68N46849, CodeNo. RK04414, roll No. 04OLE Flu A membrane: 1.5 mg/ml anti-Flu A Medix7304 antibody on test line and 1.5 mg/ml rabbit anti-mouse IgG oncontrol line striped in Nanogen POC, Toronto, Canada; Anti-Flu AFitzgerald M4090913—Europium conjugate at 1% beads concentration wasprepared in Nanogen POC, Toronto, Canada; Flu B membrane: 1.5 mg/mlanti-Flu B Medix 9901antibody on test line and 1.5 mg/ml rabbitanti-mouse IgG on control line striped in Nanogen POC, Toronto, Canada;Flu B membrane: 1.5 mg/ml anti-Flu B M2110171 antibody on test line and1.5 m mg/ml rabbit anti-mouse IgG on control line striped in NanogenPOC, Toronto, Canada; Anti-Flu B HyTest 2/3—Europium conjugate at 1%beads concentration was prepared in Nanogen POC, Toronto, Canada;Inactivated influenza A virus: Texas 1/77 (H3N2) from MicrobixBiosystems, Inc. Cat. #EL-13-02, lot #13037A3; Inactivated influenza Bvirus: Hong Kong 5/72 from Microbix Biosystems, Inc. Cat. #EL-14-03, lot#14057A2; 3× extraction buffer containing 0.15 M Tris-Cl, pH 8.0, 2.25 MNaCl, 3% BSA, 0.3% pluronic, 0.06% NaN3, 1.5% digested casein and 6 mMTCEP, was prepared in Nanogen, San Diego. Lot #2232-001; Virus dilutionbuffer: 10 mM PBS with 1% BSA; Antibody-europium conjugate dilutionbuffer: 10 mM PBS, 1% BSA and 0.2% tween-20.

Protocols. Preparation of the lateral flow test strip: Nitrocellulosemembrane striped with antibodies was laminated on a plastic card with1-2 mm overlap between the wicking pad and at the end of thenitrocellulose strip with the absorbent pad. No cover tape was appliedon any components. Strips were cut in 5 mm widths. Viruses dilution:Inactivated influenza A and influenza B positive controls were dilutedto 409.6 HA unit/mL and 40.96 HA unit/mL, respectively for assay withthe influenza test.

Antibody-Europium conjugate pretreatment: 1% Antibody-Europium conjugatewas sonicated in a bath sonicator for 4 min. An amount of conjugatedbeads was then diluted to 0.04% with antibody-europium dilution bufferand sonicated for another 4 min.

Influenza A and B test procedure with antibody Europium conjugate: To16.67 μL 3× Extraction Buffer in a test tube, add 5 μL 0.04%antibody-europium conjugate and different amount of virus. Add water toa final volume of 50 μL. Insert a lateral flow test strip in the assaysolution. Wait 15 min at room temperature. Add 50 μL 1× extractionbuffer to the same test tube. Wait for 5 min at room temperature. Placethe lateral flow test strip in a tray and measure the fluorescenceintensity. Each test condition was run with 5 replicates.

Results: FIG. 15 Analytic sensitivity of Flu A test using Europiumconjugate

TABLE 14 Statistic analysis of raw data for analytic sensitivity of FluA test using Europium conjugate Replicates HA units 1 2 3 4 5 AVG CV 0568 677 629 620 650 629  6.4% 0.021 1350 1241 1256 1308 1216 1274  4.2%0.041 1777 1535 1727 1595 1364 1600 10.2% 0.082 2171 2336 2438 2114 23362279  5.8% 0.123 4176 3745 3558 3410 3765 3731  7.7% 0.164 3989 39173963 4050 4489 4082  5.7% 0.328 8866 9979 8497 8304 7783 8686  9.5%

FIG. 16 Test the analytic sensitivity of Flu B test using Europiumconjugate and Medix 9901 on the membrane

TABLE 15 Statistic analysis of raw data for analytic sensitivity of FluB test using Europium conjugate Replicates HA units 1 2 3 4 5 AVG CV 0284 196 330 360 331 300 21.4% 0.0021 1419 1361 1572 1600 1496 1490  6.8%0.0041 1430 1467 1611 1746 1626 1576  8.1% 0.0082 2321 2520 2539 33121690 2476 23.4% 0.0123 2580 2791 2661 2737 2781 2710  3.3% 0.0164 55905111 5058 5639 5809 5441  6.2% 0.0328 8699 10903 10047 10491 9796 9987 8.4%

FIG. 17 Test the analytic sensitivity of Flu B test using Europiumconjugate and M2110171 on the membrane

TABLE 16 Statistic analysis of raw data for analytic sensitivity of FluB test using Europium conjugate HA units 1 2 3 4 5 AVG CV 0 511 501 532481 446 494 6.6% 0.0021 1368 1280 1458 1420 1395 1384 4.9% 0.0041 26972535 2708 2677 2711 2666 2.8% 0.0082 3412 3198 3817 3930 3609 3593 8.3%0.0123 6531 6560 6294 7290 6055 6546 7.1% 0.0164 6707 7328 6679 68538098 7133 8.4% 0.0328 12016 13162 12597 12907 13555 12847 4.5%

Results: Table 5-7show that the current market leader of rapid influenzaimmunoassay, the Quidel QuickVue Flu A+B test has a detection limit of4.1 HA units for influenza A Texas 1/77 and 1.6 HA units for influenza BHong Kong 5/72. When gold label is used, the present invention'sinfluenza rapid test can detect at least 0.41 HA influenza A Texas 1/77and 0.082 HA influenza B Hong 5/72 when limited assay volume and chasingbuffer were used. This is 10 and 20 times more sensitive than Quidel'sQuickVue Flu A±B in detecting influenza A Texas 1/77 and influenza BHong Kong 5/72, respectively. By applying fluorescent Europiumconjugate, the present influenza rapid test is able to detect as low as0.021 HA unit of Influenza A Texas 1/77 with a Signal/noise (S/N) ratioof 2.03 and 0.0021 HA unit of Influenza B Hong Kong 5/72 with an S/Nratio of 4.96. If a S/N ratio of 2 is considered the limit of detectionfor the Europium based assay, Flu B would be able to detect down to0.001 HA units/test. Or an improvement in sensitivity over the Quidelassay for Type B of about 1600 fold. This study clearly demonstratedthat the improvements made to nitrocellulose assay technology does yieldsignificant improvement in POC assay performance.

Example 5

Laboratory testing results to demonstrate preliminary proof of principlethat influenza H5N1 can be differentiated from seasonal human influenzaviruses using the proposed product design and instruments.

Materials: 3× Extraction Buffer containing 0.15 M Tris-Cl, pH 8.0, 2.25M NaCl, 0.3% Pluronic F68, 3% BSA, 1.5% casein and 0.06% NaN₃, lot#2232-001; 1× Extraction Buffer, lot #2232-003; 0.2 MTris(carboxyethyl)phosphine hydrochloride (TCEP), lot #2232-078;Molecular Biology Water, lot #318105; Europium dilution buffer, lot #RC6-8-06; 10 mM PBS, pH 7.2 with 1% BSA, lot #HR 7-19-06; 1% HyTest 2/3Europium conjugate with 0.025 mg antibody, lot #E2/3-270706-2; 1%M4090913 Europium conjugate with 0.025 mg antibody, lot #E13-270706-2;1% 3C8 Europium conjugate with 0.1 mg antibody, lot#E3C8-180806;Nitrocellulose membrane striped with anti-Flu A A60010044P, anti-Flu B9901, anti-H5 3G4 and rabbit anti-mouse antibody was prepared by NanogenPOC, Toronto, Canada. The antibody coating concentration was 2.0 mg/mlfor all three test lines. The antibody coating concentration for thecontrol line was 1.5 mg/ml. The distance between each test line, andtest line and control line is 5 mm±0.5 nun; Wicking pad: 0.05% tween-20treated polyester pad, 1.4 cm in width provided by Nanogen POC inToronto, Canada; Absorbent pad: Ahlstrom Grade 222 paper, 3.5 cm inwidth, purchased from Fisher, Cat. #2228-1212, lot #6150502; Inactivatedinfluenza A H1N1, Beijing/262/95 was purchased from NIBSC, 40 μg HAactivity/ampoule; Inactivated influenza A H3N2, Texas 1/77 was purchasedfrom Microbix Biosystems, Inc. lot#113037A3, 40960 HA/mL; Inactivatedinfluenza H5N1, Ck/HK/Yu22/02 from Wantai at China; Inactivatedinfluenza B, Hong Kong 5/72 was purchased from Microbix, Biosystems,Inc., lot#14057A2, 40960 HA/mL; Epstein-Barr virus purchased from ATCC,VR-1491, strain B98-8, lot #3605034

Procedures: Preparation of influenza test strips with multiple testline: Nitrocellulose membrane striped with antibodies was laminated onplastic card with 1-2 mm overlap with wicking pad and absorbent pad. Nocover tape was applied on any components. Strips were cut in 5 mm inwidth; Inactivated influenza A H3N2, Texas 1/77 and influenza B, HongKong 5/72 were diluted 10 and 100 times, respectively, with 10 mM PBS/1%BSA; Inactivated H1N1 Beijing 262/95 was reconstituted with 1 mL H₂O; 1%antibody Europium conjugate was sonicated for 4 min with Branson 1210bath sonicator. The individual conjugate was then diluted to 0.04% withEuropium conjugate dilution buffer and sonicated 3 times with 4 min eachtime in ice bath. In some experiments, all three Europium conjugateswere diluted together into the same tube. In this case, each antibodyEuropium conjugate concentration was still 0.04% bead; Assaypreparation:

For each assay, following components were added to a tube: 16.67 μL 3×Extraction Buffer; 0.5 μL 0.2 M TCEP; 5 μL diluted Europium conjugate ifall europium conjugate were diluted into the same tube or 15 μL Europiumconjugate (5 μL each if individual Europium conjugate was dilutedseparately); Virus as indicated in the figures; Adjust the final volumeto 50 μL with H₂O.

Test: Test was started by inserting the test strip into the assaysolution. After 15 min, 50 μL 1× Extraction Buffer was added to the testtube and test was carried on for another 5 min. At 20 min, the result oftest strip was read with a fluorescence reader. Each test was repeated 3times.

Results: FIG. 18 influenza rapid test strip images under UV light afterstrips were tested with buffer, influenza A subtype H3N2, H1N1 and H5N1,influenza type B and Epstein-Barr virus.

FIG. 19 Test of influenza rapid test with buffer only

FIG. 20 Test of influenza rapid test with influenza A subtype H3N2,Texas 1/77

FIG. 21 Test of influenza rapid test with influenza type B

FIG. 22 Test of influenza rapid test with influenza A subtype H5N1,Ck/HK/Yu22/02

FIG. 23 Test of influenza rapid test with influenza A subtype H1N1Beijing/262/95

FIG. 24 Test of influenza rapid test with H5N1 and influenza type B

FIG. 25 Test of influenza rapid test with Epstein-Barr virus

Results: Based on initial test results, antibodies have been selectedfor detecting influenza type A, type B and subtype H5. Strips with threedifferent test lines that detected type A, type B and subtype H5,respectively, were prepared. The results in FIGS. 1-7 show that when FluB is tested, only Flu B peak is observed. When either H1N1 or H3N2 istested, only Flu A test peak is seen and peak corresponding to subtypeH5 is not observed. Based one embodiment herein, another test linecorresponding to H1 and H3 (together) will be striped on the membraneonce anti-H1 and H3 antibodies are selected. Then another peakcorresponding to H1/H3 will be also observed in addition to Flu A peak.When subtype H5N1 was tested, both Flu A peak and H5 peak are observedwith the Flu A peak showing a stronger signal than the H5 peak. Thisresult was expected as additional optimization of MAb selection andantibody loading and other factors within the assay are optimized. Nocross-reaction of influenza test with EB virus was observed. Theseresults clearly demonstrate that the approach discovered herein canyield multiple analyte test results without interference between thetest lines.

The results shown above have clearly demonstrated that a new fullyintegrated POC technology that is highly sensitive and can independentlydetect different viral Types and subtypes on the same test strip hasbeen discovered.

Example 6 Flu B Rapid Assay Using pRNA as Capture

pRNA was fixed on the membrane to determine efficacy for capture ofcomplimentary pRNA conjugated with anti-influenza B nucleoproteinantibody, in order to generate a signal when type B virus is present inthe sample and an antibody-antigen-antibody complex is formed.

Materials: Wicking pad: 0.05% tween-20 treated polyester pad, 1.4 cm inwidth provided by Nanogen POC in Toronto, Canada; Absorbent pad:Ahlstrom Grade 222 paper, 3.5 cm in width, purchased from Fisher, Cat.#2228-1212, lot #6150502 ; Nitrocellulose Membrane: Millipore HiFlow 135membrane, 2.5 cm in width. Cat. No. SHF1350425, Lot No. R68N46849, CodeNo. RK04414, roll No. 04OLI; Nitrocellulose membrane striped with 1.5mg/ml M2110171, lot#M083106; Nitrocellulose membrane striped with 1.35mg/ml 102a4-5G4, lot#MZ-42-74247-pRNA135; Anti-Flu B HyTest 2/3—Europiumconjugate at 1% beads concentration was prepared in Nanogen POC,Toronto, Canada, lot #E2/3-270706-3; Inactivated influenza B virus: HongKong 5/72 from Microbix Biosystems, Inc. Cat. #EL-14-03, lot #14057A2;3× extraction buffer containing 0.15 M Tris-Cl, pH 8.0, 2.25 M NaCl, 3%BSA, 0.3% pluronic, 0.06% NaN3, 1.5% digested casein and 6 mM TCEP, wasprepared in Nanogen, San Diego. Lot #2232-001; 0.2 M TCEP, lot#2232-078; 1× Extraction Buffer, lot #2232-003; Europium conjugatedilution buffer, lot #RC 6-8-06; Virus dilution buffer: 10 mM PBS with1% BSA; 10 mM PBS, lot #SP09-22-06; Antibody-europium conjugate dilutionbuffer: 10 mM PBS, 1% BSA and 0.2% tween-20; Protein 5G4 for conjugationwith pRNA that would be fixed on the membrane; Anti-Flu B nucleoproteinM2110171 was purchased from Fitzgerald, Bat #578; pRNA 102a4-amn and102b4-amn (both 13 mer) were synthesized and activated by PDITC.

Protocol:

Preparation of pRNA-antibody conjugates

TABLE 17 Calculation of amounts of oligo and antibodies required forconjugation. Conjugated with Oligo/antibody Antibody Oligo AntibodyRatio required 102a4amn 76.3 nmol 5G4 10:1 1.15 mg 102b4amn 54.7 nmolM2110171  5:1 1.64 mg

Dissolve activated pRNA 102a4amn (total 229 nmol) and 102b4amn (total164 nmol) in 150 μl of water in two separate tubes.

-   -   a. Aliquot 50 μl into 3 microcentrifuge tubes for each pRNA and        dry them using a spin vac.    -   b. Place 1.5 mg of 5G4 monoclonal antibody into one spin filter        and 2.0 mg M2110171 monoclonal antibody into another spin filter        to concentrate both antibody. Antibodies were rinsed with 0.1 M        sodium borate buffer. The final concentration of 5G4 and        M2110171 were determined by the absorbance at 280 nm and were        33.35 mg/ml and 28.6 mg/ml, respectively    -   c. Take 34 μL of concentrated 5G4 (1.15 mg) and 57 μL of        concentrated M2110171. Bring the both volumes to 65 μL with 0.1        M borate buffer.    -   d. Add each antibody to the corresponding pRNA and let reaction        go for 15-20 hours at room temperature.    -   e. pRNA-antibody conjugate was then purified with a Sephadex        G-50 column with bed volume of 7 mL equilibrated with 0.01 M        PBS.

-   1. Preparation of nitrocellulose membrane striped with 102a4-5G4    -   Membrane was striped at NPOC at Toronto. See “Materials”.

-   3. Preparation of the lateral flow test strip:    -   Nitrocellulose membrane striped with antibody or 102a4-5G4 was        laminated on a plastic card with 1-2 mm overlap with wicking pad        at one end and 1-2 mm overlap with absorbent pad at the other        end. Strips were cut in 5 mm. widths.

-   4. Viruses dilution:    -   Inactivated influenza B virus was diluted to 4.096 HA unit/mL        with 0.01 M PBS and 1% BSA buffer.

Antibody Europium Conjugate Pretreatment

-   -   1% anti-Flu B 2/3-Europium conjugate was sonicated in a bath        sonicator for 4 mm. An amount of 10 μL conjugated beads was then        diluted to 0.02% with antibody-europium dilution buffer and        sonicated for 20 sec. using a probe sonicator (Fisher Model 550)        at power setting 2 for 4 pulses, 5 sec. each, with 10 sec.        incubation in ice between each pulse.

102b4-M2110171 (0.54 mg/ml after purification) was diluted to 0.1 mg/mlwith 10 mM PBS.

Assay Procedure

Take 50μL assay mixture and add it to a test tube. Insert a test stripinto the assay mixture. Wait 15 min at room temperature. Add 50 μL 1×Extraction Buffer to the same test tube and incubate for another 5minutes. Read the test strip with a LRE fluorescence reader

a. Prepare Assay Mixture for Strips with 102a4-5G4 Striped

TABLE 18 Virus HA units/test Components 0 0.002 0.004 0..008 0.016 0.0323× buffer (μL) 41.7 41.7 41.7 41.7 41.7 41.7 Water (μL) 52.1 50.8 49.647.1 42.1 32.1 0.2M TCEP (μL) 1.3 1.3 1.3 1.3 1.3 1.3 0.1 mg/ml102b4-M2110171 5.0 5.0 5.0 5.0 5.0 5.0 pRNA conj. (μL) 0.02% 2/3Europium 25.0 25.0 25.0 25.0 25.0 25.0 conj. (μL) Flu B virus 4.096 HA0.0 1.3 2.5 5.0 10.0 20.0 units/ml (μL) Total vol. for 2.5 tests (μL)125.0 125.0 125.0 125.0 125.0 125.0

b. Prepare Assay Mixture for Strips Striped with Anti-Flu B AntibodyM2110171

TABLE 19 Virus HA units/test Components 0 0.002 0.004 0..008 0.016 0.0323× buffer (μL) 41.7 41.7 41.7 41.7 41.7 41.7 Water (μL) 57.1 55.8 54.652.1 47.1 37.1 0.2M TCEP (μL) 1.3 1.3 1.3 1.3 1.3 1.3 0.02% 2/3 Europium25.0 25.0 25.0 25.0 25.0 25.0 conj. (μL) Flu B virus 4.096 HA 0.0 1.32.5 5.0 10.0 20.0 units/ml (μL) Total vol. for 2.5 tests (μL) 125.0125.0 125.0 125.0 125.0 125.0

Result

FIG. 30 A: Raw signals of test strips with 102a4-5G4 conjugate on.

TABLE 20 Net peak height of signals showed in FIG. 30A Table 16. HAunits/test 1 2 AVG Signal/Noise 0 360.0 331.9 345.9 1.0 0.002 521.1447.1 484.1 1.4 0.004 682.0 783.3 732.7 2.1 0.008 1048.5 916.2 982.4 2.80.016 1799.0 1594.3 1696.7 4.9 0.032 2745.4 3257.2 3001.3 8.7

FIG. 30B: Raw signals of test strips with antibody M2110171 on.

TABLE 21 Net peak height of signals showed in FIG. 30B HA units/test 1 2AVG signal/noise 0 155.1 142.6 148.8 1.0 0.002 495.8 538.8 517.3 3.50.004 884.2 895.9 890.0 6.0 0.008 1633.7 1698.0 1665.8 11.2 0.016 3329.42574.9 2952.2 19.8 0.032 7203.1 6974.3 7088.7 47.6

FIG. 31 Compare the sensitivity of two Flu B test strips.

The data clearly indicates that 1) pRNA conjugated with a protein, suchas 5G4 antibody in this case, can be fixed on the nitrocellulosemembrane; 2) pRNA can hybridize with a complementary oligo conjugatedwith an antibody on the nitrocellulose membrane; 3) the analyte specificantibody is still functioning after conjugated with pRNA. The result hasdemonstrated that a specific pair of pRNA can be used as a capturesystem in lateral flow rapid assay.

Example 7 Reader Europium Detection

Six different reference concentrations of Europium were used to test areader (note the following numerical references correspond to referencesutilized in figures: (1) 5×10⁻⁶ M; (5) 5×10⁻⁵ M; (3) 5×10⁻⁵M; (6)5×10⁻⁴M; (2) 5×10⁻⁴M and (4) 5×10⁻³M. Europium was encased in acrylicand different sized blocks were cut to provide different concentrations.Measurements are provided as for counts per meters ×10⁻⁴ FIG. 33.

In another example, seven (7) 1 mm wide Europium hard standards pieceswere mounted on 4 mm centers FIG. 34.

Example 8 Archive Sample

Influenza Viruses: Inactived Influenza A/Texas 1/77 (H3N2) at 40960 HAunits/mL from Microbix Biosystems, Inc. of Ontario, Canada, catalognumber EL-13-02; and inactive Influenza B/Hong Kong 5/72 at 40960 HAunits/mL from Microbix Biosystems, Inc. of Ontario, Canada, catalognumber EL-14-03.

Viral RNA Transcript Positive Controls; Influenza A RNA Control, PN:606161, LN: RH2174P73 at 50,000 copies/μL; and Influenza B RNA Control,PN: 606162, LN: RH2174P73 at 50,000 copies/μL

Extraction Buffer: 3× Extraction Buffer

The table below lists the formulation of the 3× Extraction Buffer andthe corresponding part number, lot number and vendor of each of thecomponents

TABLE 22 Catalog Component Concentration Vendor Number Lot Number TrizmaBase 150 mM Sigma T-6791 58H5431 BSA   3% SeraCare AP-4500 013-05-015Life Science Pluronic F68 0.3% Pragmatics Code 025 WPMS-561B Inc. Casein1.5% Sigma C5890 075K0108 NaN₃ 0.06%  Sigma S8032 125K2502 NaCl 2.25MSigma S7653 075K0024

Tris (2-carboxyethyl)phosphine hydrochloride (TCEP) 200 mM, made on7-6-06 by HR, powder ordered from PIERCE, Product Number: 20491, LotNumber:GL 102548

Prepare fresh 1× Extraction Buffer: Combine 200 μL of 3× ExtractionBuffer with 6 μL of 0.2 M TCEP and 394 μL of water to make 600 μL of 1×Extraction Buffer. The formulation of the1× Extraction Buffer is: 50 mMTris, 1% BSA, 0.1% Pluronic F68, 0.5% Casein, 0.02% NaN3, 0.75 M NaCland 2 mM TCEP.

Sample binding and storage materials: Whatman FTA Elute Micro Card, Cat.#WB 120401, EN: FE6231106; Whatman HalTis UNI-CORE 2.00 mm Punch, Cat.#WB640001, LN: 6023; and MicroAmp Reaction Tubes with Caps 0.2 mL, PN:N801-0540, LN: P34H4QA11, ABI

Elution materials: Distilled water, DNAse, RNAse free, Cat. #10977-015,LN: 1317578, Invitrogen

Heat source: GeneAmp PCR System 9700, ABI, QA2126

RT and PCR reagents and material:

TABLE 23 Catalog Component Vendor Number Lot Number RT Mix ProdesseGLS01 050105RM MgCl₂ 1M Ambion 9530G 064R37A MulV Reverse Transcriptase,ABI N808-0018 H03264 50 u/μL RNase Inhibitor, 20 u/μL ABI N808-0119H05526 RVA Primer Mix Nanogen Not available RH2174P100 AmpliTaq GoldPolymerase, ABI N808-0249 G03670 5 u/μL 96 Well PCR Reaction Plate ABI4306737 P03F6QA41

Materials for evaluating RT-PCR reactions: Agilent DNA 1000 Reagents:PN. 5067-1504, LN: 0623; and Agilent DNA Chips: PN 5067-1522, LN:JA21BK01

Methods

Preparation of RT Master Mix

TABLE 24 Component Volume per reaction (μL) RT Mix 11 1M MgCl₂ 0.1 MulVReverse Transcriptase (50 u/μL) 1 RNase Inhibitor (20 u/μL) 1 DNAse,RNAse Free Water 1.9 Total 15

Add 15 μL of RT master mix into a well in 96 well PCR plate

TABLE 25 Preparation of PCR Master Mix Component Volume per reaction(μL) RVA Primer Mix 39.5 AmpliTaq Gold Polymerase (5 u/μL) 0.5 Total 40

Add 40 μL of the PCR master mix into a well in 96 well plate

TABLE 26 Dilution of RNA Positive Controls: Dilute Influenza A andInfluenza B RNA with DNase, RNase free water as described in the tablebelow Start End Conc. conc. (copies/ (copies/ Dilution μL μL TotalCopies/ Remain μL) μL) Factor RNA water Volume rxn Volume 50,000 20,0002.5 8 12 20 N/A 10 20,000 2000 10 10 90 100 10000 90 2000 200 10 10 90100 1000 90 200 20 10 10 90 100 100 90 20 2 10 10 90 100 10 100

Add 5 uL of diluted RNA into proper RT reaction

TABLE 27 Dilution of Influenza A and Influenza B inactive viruses with1× Extraction Buffer μL of 1× Start Conc. End Conc. Dilution μLExtraction Total HA Remain (HA units/μL) (HA units/μL) Factor virusBuffer Volume units/disc Volume 40.96 4.096 10 5 45 50 20.5 45 4.0960.4096 10 5 45 50 2.05 45 0.4096 0.04096 10 5 45 50 0.2 45 0.040960.004096 10 5 45 50 0.02 45 0.004096 0.0004096 10 5 45 50 0.002 50

Application of samples to FTA Elute Card:

Apply 5 μL of following samples onto the sample area of the Whatman FTAElute Micro Card; 1× Extraction Buffer as a control; Virus diluted with1× Extraction Buffer; Air dry the FTA Elute Card for 15-20 min. at roomtemperature; punch out a 5 to 6 mm diameter disc with the punch from thearea that the sample was applied to then place in a 0.2 mL MicroAmpreaction tube; and

elute RNA from FTA Elute disc: Add 150 μL of water into the tubecontaining the disc and vortex 3× for 5 seconds; remove water by using asterile pipette tip; repeat addition/removal of water once; centrifugefor 5 seconds, then pipette off the excess liquid; add 50 μL of water,heat at 65° C. for 30 min. then vortex the tubes for 5 seconds; and takeout 5 μL of the liquid and add to the appropriate well of the 96 well RTplate.

RT-PCR Assay

Thermal cycler program for RT Step

Temperature (° C.) Time (minutes) Number of Cycles 22 10 1 42 60 1 95  51 4 Hold —

Transfer 10 μL of the completed RT reaction to the appropriate PCR wellcontaining PCR master mix. Pipette up and down to mix.

TABLE 28 Thermal cycler program for PCR Step Temperature (° C.) TimeNumber of Cycles 95 10 minutes 1 95 60 seconds 2 55 30 seconds 72 45seconds 94 60 seconds 38  60 30 seconds 72 30 seconds 72  7 minutes 1 4Hold —

Evaluate PCR reactions for amplicon using Agilent Bioanalyzer

RT-PCR Agilent Results: The no template control reactions did notgenerate any detectable amplicon.

RNA positive controls

TABLE 29 Influenza A Influenza B Copies/rxn band size ng/μL Pos/Rxn bandsize ng/μL Pos/Rxn 10K 233 bp 17.33 3/3 242 bp 18.28 2/2  1K 233 bp11.57 5/5 245 bp 16.96 3/3 100 233 bp  3.84 3/3 246 bp 10.62 2/2 10 [1]236 bp  0.30 3/3 246 bp  4.08 2/2

The observed band sizes of Influenza A and Influenza B RNA PositiveControls are of the correct size.

TABLE 30 Influenza Viruses HA Influenza A Influenza B units/disc bandsize ng/μL Pos/Rxn band size ng/μL Pos/Rxn 20 229 bp 17.39 2/2 246 bp12.31 2/2 2 229 bp  5.83 2/2 247 bp  3.02 2/2 0.2 230 bp  2.34 2/2 247bp  7.25 2/2 0.02 230 bp  0.34 1/2 247 bp  3.06 2/2 0.002 Not Not 0/2244 bp  1.08 2/2 detected available

Conclusion: Successfully detected RNA from lysed Influenza types A and Bviruses using the Whatman FTA Elute

Card and RT-PCR Amplification.

The assay detected down to 0.2 HA units/disc of Influenza type A virusand 0.002 HA units/disc of Influenza type B virus. The Whatman FTA EluteCard can be used to archive viral RNA for later testing in an RT-PCRassay for the detection of Influenza types A and B.

Example 9 Multi-Analyte Detection

To demonstrate proof of principle that multianalyte Influenza diagnostictest can differentiate Influenza H5N1 from seasonal flu a rapid and highsensitivity assay was developed and compared with traditional lateralflow tests using colored latex bead or colloidal gold.

A lanthanide label was utilized where latex microbeads filled withEuropium chelate, combined with a wash buffer demonstrated improvedsensitivity. Seasonal Influenza (in this example, Influenza Type B) wasclearly differentiated from avian flu H5N1 using membrane stripeddirectly with anti-Flu A, anti-Flu B and anti-H5 antibodies at differentzones on the test strip.

The data demonstrates that using pRNA and direct striping of MAb on tothe nitrocellulose as the capture systems on the same lateral flow teststrip, is able to separately detect influenza A subtype H1N1 and H5N 1as well as Type B and Type A nucleoprotein on different test zones on asingle Nitrocellulose test strip. A table summarizing the areas of theassay that were exercised in this study are is shown below.

TABLE 31 Proof of Concept Table: Assay Component DescriptionNitrocellulose membrane blocked with hydrolyzed Millipore 135 membrane,same material as currently used by casein Operations, the size isdifferent Wicking pad treated with Tween Standard Operations componentAbsorbent pad Material same as used in operations, size is differentBacking card Material same as used in operations, size is differentExtraction Buffer Designed to lyse and inactivate influenza MucolyticAgent Designed to reduce viscosity of mucus on the specimen swab WashBuffer Similar formulation to extraction buffer, designed to “wash”unbound Eu latex beads from the test strip and reduce background and“stop” the assay Type A MAb striped onto Nitrocellulose directly Showingfeasibility of directly spotting MAb in combination with pRNA Type A MAbconjugated to Europium Latex Conjugate the MAb directly to the Eumicrobeads microbeads Type B MAb Pair striped onto nitrocellulosedirectly Showing feasibility of directly spotting MAb in combinationwith. pRNA Type B MAb conjugated to Eu Microbeads See comment under TypeA above H1N1 MAb conjugated to a specific pRNA Demonstrating and showingproof of concept that pRNA oligos can be used as a generic capture agentin the assay. H1N1 MAb conjugated to Biotin Conjugate the MAb to biotinand react the conjugate with Streptavidin coated Eu beads, H5N1 MAbconjugated to a specific pRNA Demonstrating and showing proof of conceptthat pRNA oligos can be used as a generic capture agent in the assay.H5N1 MAb conjugated to Eu See comment under Type A above Europium latexmicrobeads coated with Streptavidin Developed a procedure to reactEuropium latex beads coated with and reacted with H1N1 Streptavidin witha specific MAb conjugated with biotin. After the reaction excess freebiotin is added to block the reaction sites. Europium latex microbeadscoated with Streptavidin See comment above and reacted with anti-H1 MAbEuropium latex microbeads coated with individual See comments above MAbsReader The reader will have optics better suited to the UV LED lightsource and with filters to eliminate background light emission, toimprove the performance of the system. Assay regent, single solution(mixture of extraction This will enable confirming the design with allassay reagents reagent, mucolytic agent, trehalose, buffer agents, mixedtogether. Europium latex beads with conjugated/hound detection reagentsand capture reagents with detection and capture reagents Four uniqueanalyte test lines plus a control line The assay demonstrated fourseparate functioning analyte test lines

Materials

1. Preactivated pRNAs were provided by Nanogen Bothell development team.

-   -   a. 102a10 (10 mer), lot #825-079A    -   b. 102b10 (10 mer, the complimentary pRNA of 102a10), lot        #825-065D, lot #825-065D    -   c. 3a10 (10 mer), lot #825-079C    -   d. 3b10 (10 mer, the complimentary pRNA of 3a10), lot #825-065F,        lot #825-065F

2. Antibody

-   -   a. Anti-Flu A 7304 from BiosPacific    -   b. Anti-Flu A M4090913 from Fitzgerald    -   c. Anti-Flu B 2/3 from HyTest    -   d. Anti-Flu B M2110171 from Fitzgerald    -   e. Anti-influenza H1 M322210 from Fitzgerald    -   f. Anti-influenza H1 8252K from Chemicon    -   g. Anti-influenza H5 10F7 from Xiamen University    -   h. Anti-influenza H5 3G4 from Xiamen University

3. OptiLink Carboxylate-Modified Microparticles (Europium beads)

-   -   Europium beads with 0.3 □M diameter was purchased from        ThermoFisher Scientific (Seradyn). Cat. #83470750010250,        manufacture lot #CO20512, package lot #201154.

4.Virus

-   -   Influenza B Hong Kong/5/72 from Microbix, B/Shanghai/2002 from        Dr. Bovian's laboratory, A/Taiwan/1/86 (H1N1) from NIBSC,        A/Beijing/95(H1N1) provided by Dr. Bovian's laboratory,        Bar-headed GS/QH/15/2005 (H5N1) and DK/VNM/568/2005 (H5N1) from        Dr. Guan's laboratory in Hong Kong University.

5. Three (3)× Extraction buffer containing 150 mM Tris-Cl, pH 8.0, 2.25M NaCl, 3% BSA, 1.5% digested casein, 0.3% pluronic F68 and 0.15%ProClin 300, lot #2277-038.

6. solution: 0.2 M Tris(2-carboxyethylphosphine) hydrochloride solution,lot #2277-067.

7. Recombinant Streptavidin.

8. Non-specific antibody 5G4 for conjugation with pRNA, prepared byNanogen Toronto facility and used to strip onto the nitrocellulose.

9. Strip backing card, 0.1″ super white polystyrene from G&L

10. Absorbent pad, filter paper grade 222 from Alstrom filtration

11. Wicking pad, 0.05% Tween-20 treated polyester pad.

Procedure

Preparation of Antibody-Europium Conjugate

1.1 Antibodies M4090913, 2/3, and 3G4 were conjugated with 0.3 □MEuropium beads by ThermoFisher Scientific (Seradyn).

1.2 Antibody 8252k Europium conjugate was prepared through an indirectprocedure by combining biotinylated 8252K with streptavidin coatedEuropium microparticles. Biotinylated 8252K and streptavidin Europiummicrobeads were prepared at the Nanogen Toronto facility. StreptavidinEuropium stock at 1% solid was sonicated with a bath sonicator for 5min. It was then diluted to 0.02% with Europium conjugate dilutionbuffer and sonicated with a probe sonicator for 20 s. Biotinylated 8252kwas diluted to 10 μg/ml with PBS buffer. Aliquot of 5 μL was added toeach tube. Aliquot of 5 μL 0.02% solid streptavidin Europium conjugatewas then quickly mixed with biotinylated 8252K. The conjugate wasblocked using 2.5 uL of 100 mM Biotin. The 8252k Europium conjugate wasthen directly used in the assay.

2. Preparation of antibody-pRNA conjugate: All the antibodies wereconcentrated to approximately 20 mg/ml using Microcon 50 from Amicon.The buffer was change to 0.1 M sodium borate, pH 9.0. Aliquot of 1 mgnon-specific antibody 5G4 was added to tubes containing 33.5 nmolpre-activated pRNA 102b10 or 3b10 (dry powder). Aliquot of 0.5 mgM322210 and 0.5 mg 10F7 were added to tubes containing 16.7 nmolpre-activated pRNA 102a10 and 3a10, respectively. The reaction wascarried out for 15 hours at room temperature. The antibody pRNAconjugate was purified with a Sephadex G-50 column equilibrated with 10mM PBS buffer, pH 7.0. Two peaks were resolved, the first peak withconjugate and second peak with unactivated pRNA(confirmed by HPLC, datanot shown).

3. Preparation of nitrocellulose membrane striped with four test lines.For this membrane, anti-Flu A 7304 and anti-Flu B M2110171 at 1.5 mg/mlwere striped directly for test line “A” and test line “B”, respectively.Conjugate 102b10-5G4 and 3b10-5G4, both at 0.75 mg/ml, were striped for“H1” and “H5” test line. The control line was striped with 0.5 mg/mlrabbit anti-mouse antibody. All lines were 5 mm apart. The membrane wasthen blocked with a solution containing 0.35% PVP, 0.25% Triton X-100and 0.5% digested casein, and cured at 37° C.

4. Preparation of test strip Nitrocellulose membrane striped with 4 testlines and one control line was laminated onto the backing card and has1-2 mm overlap with treated polyester pad and absorbent pad. Strips werecut into 5 mm widths using a Kinematic 2360 strip cutter.

5. Test procedure. Assay mixture was prepared as following. To a testtube, add 1.6.7 μL 3× Extraction Buffer, 0.5 μL 0.2 M TCEP, 0.1 μg102a10-M322210 conjugate, 0.1 μg 3a10-10F7 conjugate, antibody Europiumconjugate and different viruses. The final concentration of all antibodyEuropium conjugates is 0.002% solid in a final volume of 50 μL. Test wasstarted by inserting test strip into the assay mixture. After 15 min,another 50 μL of 1× Extraction Buffer was added to wash the strip.Strips were read with a fluorescence reader at 20 min.

Results.

TABLE 32 The following table compares the signal/noise ratio ofdifferent test peaks when tested with different viruses. Signal/noise ofeach test line Virus Description H5 H1 B A H5 Bar Headed 116.9 2.0 0.6245.7 GS/QH/15/2005 DK/VNM/568/2005 122.0 5.7 1.0 606.9 H1 Taiwan/1/868.5 63.8 0.4 492.7 Beijing/95 6.4 44.4 0.5 896.5 Flu B Hong Kong 5/723.5 1.3 437.1 4.8 Shanghai/2002 2.0 1.2 376.0 3.9

The data clearly shows that the test system is able to differentiatemultiple analytes. In this example, the analytes detected includedseasonal influenza subtype H1N1, Flu B from avian flu H5N1 with a goodType A signal. In this test, two pairs of pRNA were used as the capturesystem to differentiate H1 test line from H5 test line. Some lowreactivity was observed at the H1 test line position when H5 viruseswere tested indicating small amount of hybridization of pRNA 3a10conjugated on anti-H5 antibody with 102b10 striped on test line H1. Nofalse positive reaction effects were observed on the H5 specific testline when subtype H1N1 viruses were tested.

In addition, this report clearly shows that the assay, shows goodreactivity, good separation of analyte signals when a singlemulti-analyte reaction mix is used for all test specimens. In addition,the assay can detect all appropriate analytes when testing a single testspecimen at moderate viral loads simultaneously.

Conclusion: Results demonstrate that with virus of multiple subtypes andstrains significant separation of the signals for each analyte for eachviral strain or subtype tested is achieved (FIGS. 36-38). To ensure theresults had validity two separate strains of each subtype were tested.Furthermore, the results clearly demonstrate that a pRNA generic capturesystem to apply to the H1 and H5 detection systems, thus enablingutilization of the pRNA capture system for additional panels ofdifferent analytes.

Moreover, the assay can readily be adapted to incorporate additionalanalyte-specific binding agents. Further, device/assays of the inventioncan be utilized in methods of screening different analytes. For example,a large number of Anti-H3 expressing clones are can be screened todetermine.

Of course the devices/methods of the invention can be utilized to screennew viral strains (an important example is the new Fujan like strain).For example, as new MAb to new strains of the H5N1 subtype are obtained,they can be incorporated in the SCD and Test Device of the invention forscreening.

The results shown above have clearly demonstrated that a new fullyintegrated assay technology that is highly sensitive and canindependently detect different viral Types and subtypes on the same teststrip has been discovered. Further such assay can be administered in aPoint-of-Care setting.

Example 10 Multianalyte Detection: Marker Proteins

The device and methods of the invention may also be used to detectvarious analytes associated with a disease condition or nutritionalstate, or any other associative characteristics. For example, multiplecardiac markers may be detected and/or quantified:

(1) The Troponin marker: Capture Antibody is a polyclonal rabbitanti-Troponin-I and conjugated to a specific pRNA sequence (SEQ ID NO:98). Detection antibody is a mixture of two mouse MAbs and conjugated tobiotin and reacted with streptavidin coated 0.3 u Europium latexmicroparticle.

(2) The CK/MB marker. Capture antibody is a Polyclonal Goat and isconjugated to specific pRNA sequence (SEQ ID NO: 99). Detection antibodyis also a MAb and is conjugated to biotin and reacted with streptavidincoated 0.3u Europium latex microparticles.

(3) The Myoglobin marker: Capture antibody is a Rabbit anti Myoglobin.Polyclonal and is conjugated to specific pRNA sequence (SEQ ID NO: 100).Detection antibody is also a MAb and is conjugated to biotin and reactedwith streptavidin coated 0.3 u Europium latex microparticle.

(4) Control line made up of rabbit anti mouse.

Therefore, the multianlyte “point of care” assay system can quantitateeach of the markers individually in a single assay format as outlinedabove and as follows:

Using the reader with a hard Europium standard (a Europium standardlocked in acrylic and yields a uniform reading under a uniform set ofconditions over a long period of time) which ensures the reader is ableto meet fluorescent signal intensity requirements reproducibly. Thuseach analyte can be quantitated independently

Example 11 Multianalyte Detection: Viruses

As disclosed herein above, the devices of the invention can also beconfigured to detect multiple different viruses, or different subtypesof a virus. For example, the devices of the invention can be configuredto provide an influenza multianalyte plus RSV qualitative assay wheremultiple analytes can be detected from a single specimen:

(1) Influenza Type A: Capture antibody is a MAb and is conjugated tospecific pRNA sequence (SEQ ID NO: 99); Detection antibody is also a MAband is conjugated to biotin and reacted with streptavidin coated 0.3uEuropium latex microparticles

(2) Influenza Type B: Capture antibody is a monoclonal mouse MAb and isconjugated to a specific pRNA sequence (SEQ ID NO: 100); Detectionantibody is also a MAb and is conjugated to biotin and reacted withstreptavidin coated 0.3 u Europium latex microparticles.

(3) Influenza H1 and H3: Capture antibodies are MAb specific for H1 andH3 Hemagglutinin and are all conjugated to specific pRNA sequence (SEQID NO: 101); Detection antibody is also a MAb and is conjugated tobiotin and reacted with streptavidin coated 0.3 u Europium latexmicroparticles.

(4) Influenza 1-15: Capture antibody is a monoclonal mouse MAb and isconjugated to specific pRNA sequence (SEQ ID NO: 102); Detectionantibody is also a MAb and is conjugated to biotin and reacted withstreptavidin coated 0.3 u Europium latex microparticles.

(5) RSV: Capture antibody is a monoclonal mouse MAb and is conjugated tospecific pRNA sequence (SEQ ID NO: 103); Detection antibody is also aMAb and is conjugated to biotin and reacted with streptavidin coated 0.3u Europium latex microparticles.

(6) Control Line: Capture antibody is rabbit anti-mouse and binds to theMAb on the Europium latex microparticle.

Example 12 Expression of Single Chain Antibodies of 10F7 and 4D1 andTest of Their Activities

The variable region genes of the heavy and light chains of each antibodywere linked with a nucleic acid encoding a short peptide (GGGGS) to formthe DNA fragment encoding a single chain antibody. Use 10F7 VHF/10F7 VHRas the primer pair to amplify the variable region DNA fragment of 10F7heavy chain. Use 10F7 VKF/10F7 VKR as the primer pair to amplify thevariable region DNA fragment of 10F7 light chain. Use 4D1 VHF/4D1 VHR asthe primer pair to amplify the variable region DNA fragment of 4D 1heavy chain. Use 4D1 VKF/4D1 VKR as the primer pair to amplify thevariable region DNA fragment of 4D1 light chain.

Use 10F7 VHF/10F7 VKR as primers to amplify the overlapping 10F7 singlechain DNA fragment. Use 4D1 VHF/4D1 VKR as primers to amplify theoverlapping 4D1 heavy chain DNA fragment. The amplified DNA fragmentswere recovered, digested with BamH I and Sal I, and cloned intoprokaryotic expression vector pTO-T7 digested with the same restrictionenzymes. Using ER2566 E. coli as host cells, the single chain antibodyproteins were expressed using standard methods. The expressed proteinswere in the form of insoluble inclusion bodies. The inclusion bodieswere broken up by ultrasound treatment, and the resulting sediments werepurified using standard methods. The purified sediments were dissolvedin 8M urea. The urea solution was dialyzed slowly in 1× PBS solution,centrifuged at 12000 rpm for 10 min to remove the remaining sediments.The final purified single chain antibody solution was tested foractivities.

Select 26 strains of H5N1 viruses to test the activities of the abovepurified 10F7 and 4D1 single chain antibodies using HI assay asdescribed above. The concentration of 10F7 single chain antibody is usedat 1.06 mg/ml. The concentration of 4D1 single chain antibody is used at0.34 mg/ml. The 4D1 single chain antibody exhibits HA inhibitionactivity against 23 of the virus strains. The 10F7 single chain antibodyshows HA inhibition activity against 14 of the virus strains (Table 33below).

TABLE 33 HA inhibition activities of the three single chain antibodiesagainst the 25 H5N1 viruses. H5N1 ScFv Virus Strains 4D1 10F7 8H5A1 >8 >8 3.5 A2 >8 >8 4.5 A3 >8 >8 3.5 A5 >8 >8 4 A6 7 7.5 3 A7 6 6.5 3A8 >8 6 2.5 B1 >8 7 4 B2 0 0 0 B3 >8 >8 5 B4 >8 >8 5 B5 0 0 0 B6 >8 73.5 B7 >8 7 4 B8 >8 7 3 C2 >8 2 1 C3 >8 1 0 D1 >8 2.5 2.5 D2 7.5 2.5 2E1 1 0 0 E2 1 0 0 F2 5 0 0 F3 3 0 0 G1 3.5 0 0 H1 6 <1 0 H2 4 0 0

HI titer is diluted by the “n”th power of 2. “n” is the numbers shown inthe table.

The activity of the above purified 10F7 single chain antibody was testedusing the neutralization method. 7 virus strains that were isolated fromchicken, duck and various wild birds in Hong Kong, Indonesia, Qinghaiand other areas during the period from 2002 to 2006 were used to testthe activity of 10F7 using the HI assay. The antibody showed goodneutralization activity against 5 of the virus strains (Table 34). At 64times of dilution, the antibody was still able to inhibit virusinfection of host cells.

TABLE 34 10F7 single chain antibody neutralization test results. Virusstrain dilution of 10F7 scFv CK/HK/Yu22/02 64 DK/IDN/MS/04 16CK/IDN/2A/04 32 BhGs/QH/15/05 16 CK/HK/213/03 <1 CP Heron/HK/18/05 8Oriental Magpie Robin/HK/366/2006 <1

Example 13 Detection of 7aa Peptides Activities

The three bacteriophages containing the 7aa peptides of 8H5A, 8H5E and3C8A were amplified in large numbers. They were dissolved in PBS afterbeing precipitated with PEG. Phage titer was between 10¹¹ and 10¹².Microplates were pre-coated with monoclonal antibodies 8H5, 4A1, 9N7 and4D11 at 5 μg/ml. The plates were blocked with PBS containing 5% milk.The three bacteriophages were serially diluted; and added to the plates.The reaction was carried on for 1 hr. Then the plates were washed for 5times. 1:5,000 diluted mouse anti-M13/HRP antibody (Amersham PhamarciaBiotech, UK) was added as the secondary antibody and incubated for 0.5hr. The results were read after the reaction was completed. The resultsare shown in Table 35 below, which demonstrated that the specificreactions between the peptide 8H5A and the monoclonal antibody 8H5 weregood, and the specific reactions between 8H5A and the other threemonoclonal antibodies were weak. The specific reaction between 8H5E andmonoclonal antibody 8H5 was relatively poor.

TABLE 35 Detection results of the specific binding activity of 7aaPeptides to monoclonal antibodies Monoclonal Antibody 8H5A (1:1000) 8H5E(1:1000) 8H5 0.559 0.25 4A1 0.158 0.142 9N7 0.062 0.065 4D11 0.118 0.078

Example 14 MAB Capture System vs. pRNA Capture System Using NanogenInfluenza A Test

To compare the sensitivity of Nanogen Influenza A Test using 2 differentcapture systems and to compare the pRNA capture system using 2 differenttest strips

Procedure for cutting test strips: Prepare the membrane card by placingabsorbent pad (3.5 cm wide) and Tween-20 pre-treated; precut polyesterwicking pad (1.4 cm wide) on the adhesive side with 1 mm overlap withthe membrane, striped with appropriate antibody. Cut this prepared cardinto 5 mm wide test strips with a paper cutter

Virus Dilution: Dilute Influenza A/Texas/1/77 40960 HA units/mL viruswith 1% BSA in PBS according to the following dilution scheme.

TABLE 36 Dilution Scheme for Flu A Virus 1% BSA in End End Start conc.dilution Start vol. PBS vol. vol. conc. (HA (HA units/mL) factor (μL)(μL) (μL) units/mL) 40960  1:100 1 99 100 409.6 409.6 1:10 50 450 50040.96 40.96 1:10 20 180 200 4.096

Europium Dilution: Dilute 17-10-06-1 europium conjugate to 0.02% usingthe europium dilution buffer; 245 μL of europium dilution buffer+5 μL of1% stock. Sonicate the diluted europium conjugate using a probesonicator (Fischer Model 550) at power setting of 3, total time 20seconds (10 sec on time and 5 sec off time).

pRNA Conjugate dilution: Dilute 102a10-7304 (0.39 mg/ml) conjugate to0.1 mg/ml with 10 m.M PBS, pH 7.2; 37.2 μL of PBS+12.8 μL of 102a10-7304conjugate.

Assay Procedure: Prepare the assay mixtures according to the followingtables

TABLE 37 Assay Mix for the MAB capture system 40.96 HA 4.096 HA units/mlunits/ml HA units/test 0 0.01 0.02 0.04 0.1 1 3× extraction 41.75 41.7541.75 41.75 41.75 41.75 buffer (μL) 0.2M TCEP (μL) 1.25 1.25 1.25 1.251.25 1.25 0.02% europium 12.5 12.5 12.5 12.5 12.5 12.5 conj. (μL) Flu Avirus (μL) 0 6.25 12.5 25 6.25 62.5 water (μL) 69.5 63.25 57 44.5 63.257 Total vol. for 125 125 125 125 125 125 2.5 test (μL)

TABLE 38 Assay Mix for the pRNA capture system 4.096 HA units/ml 40.96HA units/ml HA units/test 0 0.01 0.02 0.04 0.1 1 3× extraction 83.5 83.583.5 83.5 83.5 83.5 buffer (μL) 0.2M TCEP (μL) 2.5 2.5 2.5 2.5 2.5 2.50.02% europium 25.0 25.0 25.0 25.0 25.0 25.0 conj. (μL) Flu A virus (μL)0.0 12.5 25.0 50.0 12.5 125.0 0.1 mg/mL 102a10- 5.0 5.0 5.0 5.0 5.0 5.07304 conj. (μL) water (μL) 134.0 121.5 109.0 84.0 121.5 9.0 Total vol.for 250.0 250.0 250.0 250.0 250.0 250.0 5 test (μL)

TABLE 39 Materials Material Lot Note 1 Absorbent Pad: Grade 222, 12 IN ×12 6150502 cat # 2228-1212 IN 2 Wicking Pad: Tween 20 Pre-treated06PPP0157C Grade 6613 Polyester Pad 3 Anti-flu A Medix 7304 (1.5 mg/ml)310806 membrane 4 102b10-5G4 striped membrane 131206-2-1 conc. 0.75mg/ml, w/o blocking 5 102b10-5G4 striped membrane 131206-2-2 conc. 0.75mg/ml/w/ 0.5% casein (toronto) blocked 6 3x Extraction Buffer 2271-001DOM Nov. 15, 2006 7 1x Extraction Buffer 2232-003 DOM Nov. 15, 2006 80.2M TCEP 2232-180 DOM Nov. 9, 2006 9 Molecular Biology Water 1347929CAT # 10977-015, GIBCO 10 1% BSA in 0.10M Phosphate Buffered 2271-036DOM Dec. 14, 2006 Saline 11 Flu A/Texas/1/77 virus @ 40960 HA 13037A3CAT # EL-13-02 units/mL 12 Europium dilution buffer N/A DOM Jun. 8, 2006by Roy Chung 13 Europium conjugate with M4090913 17-10-06-1 Prepared byPhilip Lam 14 102a10-7304 pRNA conjugate 2271-028-2 DOM Dec. 8, 2006,conc. 0.39 mg/ml 15 10 mM PBS pH 7.2 2271-035 DOM Dec. 14, 2006

Incubate 7304 (1.5 mg/mL) test strips with MAB CAPTURE SYSTEM assay mix.

Incubate 102b1.0-5G4 (0.75 mg/mL) w/o blocking, and w/0.5% caseinblocked test strips with pRNA CAPTURE SYSTEM assay mix.

Run the experiment in duplicates.

Let it stand at room temperature for 15 min.

Add 50 μL of 1× extraction buffer to the same tube to wash the teststrips.

After 5 min, read the test strips using a fluorescence reader.

Results

TABLE 40 MAB capture system (See FIG. 39) HA units/test 0 0.01 0.02 0.040.1 1 1 528.1 793.7 935.7 1421.6 2550.1 21111.3 2 569.6 776.0 1037.51440.0 2395.4 19961.8 AVG 548.8 784.9 986.6 1430.8 2472.8 20536.5 S/N1.4 1.8 2.6 4.5 37.4

TABLE 41 pRNA capture system with 0.75 mg/ml membrane blocked with 0.5%casein (See FIG. 40) HA units/test 0 0.01 0.02 0.04 0.1 1 1 114.6 434.1703.0 1360.9 3222.7 21209.1 2 222.6 628.5 804.6 1385.1 2891.4 19440.1AVG 168.6 531.3 753.8 1373.0 3057.1 20324.6 S/N 3.2 4.5 8.1 18.1 120.5

TABLE 42 pRNA capture system with 0.75 mg/ml membrane without blocking(See FIG. 41) HA units/test 0 0.01 0.02 0.04 0.1 1 1 284.4 737.9 940.01880.4 3336.0 22387.5 2 318.1 612.2 814.6 1471.3 3560.8 22383.6 AVG301.3 675.1 877.3 1675.9 3448.4 22385.6 S/N 2.2 2.9 5.6 11.4 74.3

Conclusion: As the data indicates the sensitivity of the test is highestwith pRNA capture system when used with test strips blocked with 0.5%casein from Toronto with a single to noise ration of 120 with 1 HA unitbut using direct spotting of MAb onto the test strip the S/N ratio wasonly 37.4 indicating the pRNA capture system significantly improved thesensitivity of the assay system.

8H5 Vh Nucleotide sequence SEQ ID NO: 1 caggttcagc tgcagcagtc tggagctgagctgatgaagc ctggggcctc agtgaagata tcctgcaagg ctactggcta cactttcagtaactactgga tagagtggat aaagcagagg cctggacatg gccttgagtg gattggagagattttacctg gaagcgatag aacaaactac aatgggaagt tcaagggcaa ggccacattcactgcagata catcctccaa cacagcccac atgcaactca gtagcctgac atctgaggactctgccgtct attactgtgc aaatagatac gacgggtatt attttggttt ggattactggggtcaaggaa cctcagtcgc cgtctcctca gcc SEQ ID NO: 2 Gln Val Gln Leu GlnGln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala1               5                   10                  15 Ser Val LysIle Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr            20                  25                  30 Trp Ile Glu TrpIle Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile        35                  40                  45 Gly Glu Ile Leu ProGly Ser Asp Arg Thr Asn Tyr Asn Gly Lys Phe    50                  55                  60 Lys Gly Lys Ala Thr PheThr Ala Asp Thr Ser Ser Asn Thr Ala His65                  70                  75                  80 Met GlnLeu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                85                  90                  95 Ala Asn ArgTyr Asp Gly Tyr Tyr Phe Gly Leu Asp Tyr Trp Gly Gln            100                 105                 110 Gly Thr Ser ValAla Val Ser Ser Ala 8H5 Vk Nucleotide sequence SEQ ID NO: 3 gaaatcgtgctcacccagtc tccagcaatc atgtctgcat ctctagggga gaaggtcacc atgagctgcagggccagctc aagtgtaaat ttcgtttact ggtaccagca gaggtcagat gcctcccccaaactattgat ttactattca tccaacctgg ctcctggagt cccacctcgc ttcagtggcagtgggtctgg gaactcttat tctctcacaa tcagcggctt ggagggtgaa gatgctgccacttattactg ccagcacttt actagttccc cgtacacgtt cggagggggg accaacctggaaataaaacg g 8H5 Vk Amino Acid sequence SEQ ID NO: 4 Glu Ile Val Leu ThrGln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly1               5                   10                  15 Glu Lys ValThr Met Ser Cys Arg Ala Ser Ser Ser Val Asn Phe Val            20                  25                  30 Tyr Trp Tyr GlnGln Arg Ser Asp Ala Ser Pro Lys Leu Leu Ile Tyr        35                  40                  45 Tyr Ser Ser Asn LeuAla Pro Gly Val Pro Pro Arg Phe Ser Gly Ser    50                  55                  60 Gly Ser Gly Asn Ser TyrSer Leu Thr Ile Ser Gly Leu Glu Gly Glu65                  70                  75                  80 Asp AlaAla Thr Tyr Tyr Cys Gln His Phe Thr Ser Ser Pro Tyr Thr                85                  90                  95 Phe Gly GlyGly Thr Asn Leu Glu Ile Lys Arg             100                 105 3C8Vh Nucleotide sequence SEQ ID NO: 5 cagatccagt tggtgcagtc tggacctgagctgaagaagc ctggagagac agtcaagatc tcctgcaagg cctctgggta cagcttcacaaactatggaa tgaactgggt gaagcaggct ccaggaaagg gtctaaagtg gatgggctggataaacacct acaccggaga gccagcctat gctgatgact tcaagggacg gtttgccttctctctggaaa cctctgccag cactgcctat ttgcagatca acaacctcaa aaatgaggacacggctacat atttctgtgc aagatggaat agagatgcta tggactactg gggtcaaggaacctcggtca ccgtatctag c 3C8 Vh Amino Acid sequence SEQ ID NO: 6 Gln IleGln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1               5                   10                  15 Thr Val LysIle Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr            20                  25                  30 Gly Met Asn TrpVal Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met        35                  40                  45 Gly Trp Ile Asn ThrTyr Thr Gly Glu Pro Ala Tyr Ala Asp Asp Phe    50                  55                  60 Lys Gly Arg Phe Ala PheSer Leu Glu Thr Ser Ala Ser Thr Ala Tyr65                  70                  75                  80 Leu GlnIle Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys                85                  90                  95 Ala Arg TrpAsn Arg Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser            100                 105                 110 Val Thr Val SerSer         115 3C8 VK Nucleotide sequence SEQ ID NO: 7 gacattgtgctgacccaatc tccagcttct ttggctgtgt ctcttgggca gagggccacc atatcctgcagagccagtga aagtgttgat agttctgaca atagtcttat gcactggtac cagcagaaaccaggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct gggatccctgccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat cctgtggaggctgatgatgt tgcaacctat tactgtcagc aaagtattgg ggatcctccg tacacgttcggaggggggac caagctggaa ataaaacgg 3C8 VK Amino Acid sequence SEQ ID NO: 8Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly1               5                   10                  15 Gln Arg AlaThr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Ser            20                  25                  30 Asp Asn Ser LeuMet His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro        35                  40                  45 Lys Leu Leu Ile TyrArg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala    50                  55                  60 Arg Phe Ser Gly Ser GlySer Arg Thr Asp Phe Thr Leu Thr Ile Asn65                  70                  75                  80 Pro ValGlu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Ile                85                  90                  95 Gly Asp ProPro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg            100                 105                 110 10F7 VhNucleotide sequence SEQ ID NO: 9 caggtccaac tgcagcagcc tggggctgaacttgtgaagc ctggggcttc agtgaagctg tcctgcaagg cttctggcta caccttcaccagctactgga tgcactgggt gaagcagagg cctggacagg gccttgagtg gatcggagagattgatcctt ctgattctta tactaactac aatcagaagt tcaagggcaa ggccacattgactgtagaca aatcctccag cacagcctac atgcagctca gcagcctgac atctgaggactctgcggtct attactgtgc aagggggggt acaggagact ttcactatgc tatggactactggggtcaag gcacctcggt caccgtatca tcg 10F7 Vh Amino Acid sequence SEQ IDNO: 10 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala1               5                   10                  15 Ser Val LysLeu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr            20                  25                  30 Trp Met His TrpVal Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile        35                  40                  45 Gly Glu Ile Asp ProSer Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe    50                  55                  60 Lys Gly Lys Ala Thr LeuThr Val Asp Lys Ser Ser Ser Thr Ala Tyr65                  70                  75                  80 Met GlnLeu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                85                  90                  95 Ala Arg GlyGly Thr Gly Asp Phe His Tyr Ala Met Asp Tyr Trp Gly            100                 105                 110 Gln Gly Thr SerVal Thr Val Ser Ser         115                 120 10F7 VK Nucleotidesequence SEQ ID NO: 11 gacatcctga tgacccaatc tccatcctcc atgtctgtatctctgggaga cacagtcagc atcacttgcc atgcaagtca gggcattagc agtaatatagggtggttgca gcagaaacca gggaaatcat ttaagggcct gatctatcat ggaaccaacttggaagatgg agttccatca aggttcagtg gcagtggatc tggagcagat tattctctcaccatcagcag cctggaatct gaagattttg cagactatta ctgtgtacag tatgttcagttcccgtacac gttcggaggg ggcaccaagc tggaaatcaa acgg 10F7 VK Amino Acidsequence SEQ ID NO: 12 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met SerVal Ser Leu Gly1               5                   10                  15 Asp Thr ValSer Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser Asn            20                  25                  30 Ile Gly Trp LeuGln Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile        35                  40                  45 Tyr His Gly Thr AsnLeu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly    50                  55                  60 Ser Gly Ser Gly Ala AspTyr Ser Leu Thr Ile Ser Ser Leu Glu Ser65                  70                  75                  80 Glu AspPhe Ala Asp Tyr Tyr Cys Val Gln Tyr Val Gln Phe Pro Tyr                85                  90                  95 Thr Phe GlyGly Gly Thr Lys Leu Glu Ile Lys Arg             100                 105Artificial sequence/Unknown Organism SEQ ID NO: 13. catgggatgctgccggtgta t Artificial Sequence/Unknown Organism SEQ ID NO: 14.aattctgggc cttggctgac g Artificial Sequence/Unknown Organism SEQ ID NO:15. tggccgcctc tgtcgaagaa g 4D1 VH Nucleotide sequence SEQ ID NO: 16.caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaacctgtcctgtaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagaggcctggacaag gccttgagtg gatcggagag attgatcctt ctgatagttt tactacctacaatcaaaact tcaaagacag ggccacattg actgtagaca aatcatccag cacagcctacatgcagctca gaagtctgac atctgaggac tctgcggtct attactgtgc cagggggggtccaggagact ttcgctatgc tatggattac tggggccaag gcacctcggt caccgtctcc tca4D1 VH Amino Acid sequence SEQ ID NO: 17 Gln Val Gln Leu Gln Gln Pro GlyAla Glu Leu Val Lys Pro Gly Ala1               5                   10                  15 Ser Val AsnLeu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr            20                  25                  30 Trp Met His TrpVal Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile        35                  40                  45 Gly Glu Ile Asp ProSer Asp Ser Phe Thr Thr Tyr Asn Gln Asn Phe    50                  55                  60 Lys Asp Arg Ala Thr LeuThr Val Asp Lys Ser Ser Ser Thr Ala Tyr65                  70                  75                  80 Met GlnLeu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                85                  90                  95 Ala Arg GlyGly Pro Gly Asp Phe Arg Tyr Ala Met Asp Tyr Trp Gly            100                 105                 110 Gln Gly Thr SerVal Thr Val Ser Ser          115                 120 4D1 VK Nucleotidesequence SEQ ID NO: 18 gacatcctga tgacccaatc tccatcctcc atgtctgtatctctgggaga cacagtcagc atcacttgcc atgcaagtca gggcattagc agtaatatagggtggttgca gcagaaacca gggaaatcat ttaagggcct gatctatcat ggaaccaacttggaagatgg agttccatca aggttcagtg gcagtggatc tggagcagat tattctctcaccatcagcag cctggaatcc gaagactttg cagactatta ctgtgtacag tatgttcagtttccctacac gttcggaggg gggaccaagc tggaaataaa acgggct 4D1 Vk Amino Acidsequence SEQ ID NO: 19 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met SerVal Ser Leu Gly1               5                   10                  15 Asp Thr ValSer Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser Asn            20                  25                  30 Ile Gly Trp LeuGln Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile        35                  40                  45 Tyr His Gly Thr AsnLeu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly    50                  55                  60 Ser Gly Ser Gly Ala AspTyr Ser Leu Thr Ile Ser Ser Leu Glu Ser65                  70                  75                  80 Glu AspPhe Ala Asp Tyr Tyr Cys Val Gln Tyr Val Gln Phe Pro Tyr                85                  90                  95 Thr Phe GlyGly Gly Thr Lys Leu Glu Ile Lys Arg Ala            100                 105 3G4 VH Nucleotide sequence SEQ IDNO: 20 caggtccaac tgcagcagtc tggggctgag ctggtgaggc ctggggtctc agtgaagatttcctgcaagg gttctggcta cacattcact gattatgcta tgcattgggt gaagcagagtcatgcaaaga gtctagagtg gattggactt attaatactg actatggtga tactacttacaaccagaagt tcaagggcaa ggccacaatg actgtagaca aatcctccaa cacagcctatatggaacttg ccagactgac atctgaggat tctgccatct attactgtgc aagatcggactatgattact atttctgtgg tatggactac tggggtcaag gaaccacggt caccgaatct cta3G4 VH Amino Acid sequence SEQ ID NO: 21 Gln Val Gln Leu Gln Gln Ser GlyAla Glu Leu Val Arg Pro Gly Val1               5                   10                  15 Ser Val LysIle Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr            20                  25                  30 Ala Met His TrpVal Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile        35                  40                  45 Gly Leu Ile Asn ThrAsp Tyr Gly Asp Thr Thr Tyr Asn Gln Lys Phe    50                  55                  60 Lys Gly Lys Ala Thr MetThr Val Asp Lys Ser Ser Asn Thr Ala Tyr65                  70                  75                  80 Met GluLeu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys                85                  90                  95 Ala Arg SerAsp Tyr Asp Tyr Tyr Phe Cys Gly Met Asp Tyr Trp Gly            100                 105                 110 Gln Gly Thr ThrVal Thr Glu Ser Leu         115                 120 2F2 VH Nucleotidesequence SEQ ID NO: 24 caggtgcagc tgaaggagtc aggacctggc ctggtggcgccctcacagcg cctgtccatc acatgcaccg tctcagggtt ctcattaacc ggctatggtgtacactggat tcgccagtct ccaggaaagg gtctggagtg gctgggaatg atatgggctgagggaagaac cgactataat tcagttctca aatccagact gagcatcaat aaggacaattccaggagcca agttttctta gaaatgaaca gtctgcaaac tgatgacaca gccaggtactactgtgccag agaggtgatt actacggaag cctggtactt cgatgtctgg ggccaaggaacctcggtcac cgaatct 2F2 VH Amino Acid sequence SEQ ID NO: 25 Gln Val GlnLeu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1               5                   10                  15 Arg Leu SerIle Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr            20                  25                  30 Gly Val His TrpIle Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu        35                  40                  45 Gly Met Ile Trp AlaGlu Gly Arg Thr Asp Tyr Asn Ser Val Leu Lys    50                  55                  60 Ser Arg Leu Ser Ile AsnLys Asp Asn Ser Arg Ser Gln Val Phe Leu65                  70                  75                  80 Glu MetAsn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala                85                  90                  95 Arg Glu ValIle Thr Thr Glu Ala Trp Tyr Phe Asp Val Trp Gly Gln            100                 105                 110 Gly Thr Ser ValThr Glu Ser         115 2F2 VK Nucleotide sequence SEQ ID NO: 26gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctctctttcctgca gggccagcca gagtattagc gactacttat actggtatca acaaaaatcacatgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctccagattcagtg gcagtggatc agggtcagat ttcactctca ctatcaacag tgtggaacctgaagatgttg gaatgtatta ctgtcaaaat ggtcacacct ttccgctcac gttcggtgctggcaccaagc tggaaatcaa acgg 2F2 VK Amino Acid sequence SEQ ID NO: 27 AspIle Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1               5                   10                  15 Asp Arg ValSer Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr            20                  25                  30 Leu Tyr Trp TyrGln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile        35                  40                  45 Lys Tyr Ala Ser GlnSer Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly    50                  55                  60 Ser Gly Ser Gly Ser AspPhe Thr Leu Thr Ile Asn Ser Val Glu Pro65                  70                  75                  80 Glu AspVal Gly Met Tyr Tyr Cys Gln Asn Gly His Thr Phe Pro Leu                85                  90                  95 Thr Phe GlyAla Gly Thr Lys Leu Glu Ile Lys Arg.

1.-71. (canceled)
 72. A method for detection of one or more analytes ina fluid sample comprising: (a) contacting a fluid sample with a systemcomprising; (i) a sample collection implement comprising (1) a swab, (2)a chamber containing fluid for extracting said swab, (3) a plurality ofpairs of first and second detection reagents, each pair capable ofspecifically binding a different target analyte in said fluid sample;wherein either said first or second detection reagents further comprisesa capture moiety; (ii) a test device comprising a substrate defining alateral flow path comprising a plurality of test regions positionedwithin said flow path, said test regions each containing an immobilizedreagent capable of specifically binding to said capture moiety, whereineach of the test regions comprises a different immobilized reagentcapable of binding a different capture moiety; and at least one controlregion disposed in said lateral flow path, wherein said control regionpermits a determination of proper performance of the sensor; (b)detecting whether one or more analytes bind to said immobilizedcapturing reagent, disposed in said test device; and (c) determiningwhether one or more analytes are present in said fluid sample, if asignal is detected in at least one of said plurality of test regions.73. The method of claim 72, wherein said sample collection implementfurther comprises a reservoir for preserving a portion of said sample.74. The method of claim 72, where said result remains stable forvisualization for a time period greater than 30 minutes.
 75. The methodof claim 72, wherein said system's sensitivity, based on dilution, isequivalent to 2500 copies of influenza nucleic acid and specificity is98% or greater.
 76. The method of claim 72, wherein said immobilizedreagent is selected from the group consisting of an oligonucleotide,avidin, streptavidin, pRNA, aptamer or a combination thereof.
 77. Themethod of claim 72, wherein said immobilized capturing agent is pRNA,78. The method of claim 72, wherein each reagent in said plurality offirst detection reagents comprises a label.
 79. The method of claim 78,wherein said label is selected from a group consisting of a fluorophore,a chromophore, a microparticle, a metal or a combination thereof. 80.The method of claim 78, wherein said label is europium
 81. The method ofclaim 72, wherein said multiple one or more analytes are one or moreviruses.
 82. The method of claim 81, wherein said one or more virusesinclude influenza A and/or influenza B strain.
 83. The method of claim81, wherein said one or more viruses include different virus strains anddifferent virus subtypes.
 84. The method of claim 83, wherein saiddifferent strains or said different subtypes include, pandemic virus, anon-pandemic virus, vaccine-available virus, vaccine-unavailable vims,or a combination thereof.
 85. The method of claim 72, wherein each ofsaid plurality of test regions comprises said immobilized reagentspecific for said capture. 86.-154. (canceled)
 155. The method of claim72, wherein the first and second detection reagents are separate orcombined together in a pellet, a powder, a pill, a bead, a pressedlyophilized powder, or are dried on a solid support.
 156. The method ofclaim 72, wherein the first and second detection reagents are in soliddried form.